Cate, 19, who had already booked her ticket by coming runner-up in the 100 freestyle earlier this week, edged out her 17-year-old sister Bronte to win the title in 24.44 seconds at the South Australia Aquatic and Leisure Centre.
Australia's last siblings to qualify were Karen and Narelle Moras and brothers Neil and Greg Rogers at the 1972 Munich Games.
The Campbell pair ensured triple Olympic champion Libby Trickett missed out on an individual berth for London, although she had qualified for the 100 freestyle relay team on Tuesday.
"I can't be happier, it's just been an incredible, incredible journey," said world bronze medallist Cate, who also won bronze in the event on her Olympic debut as a 16-year-old at the Beijing Games.
"I'm just so glad that I get to stay in London with my best friend, it's just awesome."
Jarrod Poort sprinted away from Mack Horton in the last lap to win the men's 1,500 freestyle but missed out on automatic qualification to London with his time 1.5 seconds slower than the Olympic A standard of 15 minutes, 11.83 seconds.
Ben Treffers and Leiston Pickett won the non-Olympic men's 50 backstroke and the women's 50 breaststroke titles respectively on the last night of the national trials.