Seebohm came agonisingly close to breaking the world record when she won her heat in 58.23 seconds, just 0.11 outside the world record set by Britain's Gemma Spofforth at the 2009 World Championships in Rome. Her time was enough to set a new Olympic record.
Franklin, the rising star of the American women's team, was second fastest overall in 59.37 with Australia's Belinda Hocking third in 59.61. Spofforth finished 12th to safely advance to Sunday night's semi-finals.
"You can't take any chances, we are at the Olympics," Franklin said. "Emily's race was unbelievable but I have no control over her and what she does. The only thing I can control is myself."
The final will be held on Monday with Seebohm looming as the favourite after saying she can swim even faster.
"It was not the hardest I can go, but you never know what everyone else will do," she said.
"The time that I did was incredible. It just shows I wasn't at my best (last year)."
Sun Yang and Ryan Lochte provided a sneak preview of what now looms as the most anticipated race of the Olympic swimming competition when they set the fastest qualifying times for the men's 200 metres freestyle.
Swimming in the lanes next to each other, the pair treated their heat like a game of cat and mouse, eyeballing each other over the first three and a half laps before shifting up another gear in the last 25 metres.
Sun, who won the 400 freestyle gold medal for China the previous night, got his hand on the wall first in a time of one minute 46.24 seconds with Lochte a close second.
But neither man was trying their hardest as they try to manage their busy programmes. Lochte was in hot demand after he demolished his opponents, including Michael Phelps, to win the 400 individual medley on Saturday.
"The 400 IM took a lot out of me last night but it is a new day," Lochte said. "I didn't get to bed until 2am."
South Korea's Park Tae-Hwan was fifth fastest. He finished runner-up to Sun in the 400 free after being disqualified in the heats but winning an appeal to be reinstated.
Germany's world record holder Paul Biedermann was 10th. Phelps, who won the race in Beijing four years ago, did not enter this time despite winning the event at the US trials.
Lithuanian teenager Ruta Meilutyte provided her older and more experienced Olympic rivals with an ominous warning of her intentions when she topped the qualifiers of the women's 100 metres breaststroke heats.
The 15-year-old, who was competing at European Youth events just a year ago, showed no signs of nerves on the big stage as she easily won her heat in one minute 05.56 seconds.
Australia's Leisel Jones, the defending Olympic champion who won a silver medal at the 2000 Sydney Games when she was 15, finished second in the heat and fifth overall.
"I am pretty happy with that - probably one of the best heat swims I have done," said Jones. "You can't afford to relax. Most of the girls have gone pretty hard this morning. You can't afford to go easy and miss the semis."
American Rebecca Soni, the world champion and favourite to win the gold, was second overall in 1:05.75.
"It feels good to finally get a race in," said Soni. "I am not focusing on any pressure, or on defending any titles. I am just going to have fun and race."
Soni said she was impressed by Meilutyte.
"It's great to see when someone swims faster than they thought they could," said Soni. "That joy rubs off on the rest of us."
American Matt Grevers posted the fastest qualifying time for the men's 100 metres backstroke.
Grevers, who was runner-up to his countryman Aaron Peirsol at the Beijing Games, steamed to victory in 52.92 seconds, conserving plenty of energy for the 4x100 freestyle relay heats, which were held in the same session.
China's Cheng Feiyi was second overall while Grevers' compatriot Nick Thoman was third fastest. World champion Camille Lacourt of France was fourth.
Lacourt's joint world champion Jeremy Stravius, the pair dead heated in Shanghai, did not qualify for the Olympics after finishing third at the French trials earlier this year.
Grevers is the latest in a long line of great American backstrokers but spent most of his career in the shadows of Peirsol before succeeding his mantle when he retired.
The Australian men's 4x100 metres freestyle relay team have been demolishing their opponents for the past year and now they are on course to win the Olympic gold medal.
Despite not racing with their best four swimmers, the Australians still topped the qualifiers during Sunday's heats, stopping the clock at three minutes, 12.29 seconds.
But they did give an insight into how strong they were when they unleashed their biggest gun, James Magnussen.
Dubbed the "missile" by Australian media, Magnussen showed why he is the favourite to win the individual 100 freestyle when he produced the fastest split time of the morning, 47.35 seconds, but still looking like he was having a weekend paddle.
In the lane next to him was Jason Lezak, remembered forever for his stunning anchor leg in Beijing that ultimately helped Michael Phelps win his eight gold medals.
Lezak dived in almost half a second before Magnusson, a big margin for elite sprinters, but finished 0.30 behind as the U.S. qualified second fastest overall.
Qualified countries can change their lineups for the final but the makeup of the U.S. team remains uncertain with Michael Phelps certain to be added and possibly Ryan Lochte.
"There could be a lot of politics but our team is pretty mature - we understand the coaches probably know best," said Matt Grevers.
"I believe they will put the best possible relay out there. If that includes me that's fine, I'm pumped.
"You have to play with what is hot and Ryan Lochte is a good hand right now."
Russia finished third and France were fourth.
France's Alain Bernard, the individual 100 champion in Beijing, is retiring after the Olympics after only qualifying for the relay.
"It is a special moment as it is my last day as a high-level athlete, so it has a special flavour," he said.
"But leaving emotions aside, the job this morning was to qualify for the final and out of the four that swam this morning, only two will swim this evening. We are not yet sure who."
- Michael Phelps
- Ryan Lochte