Sablikova clocked six minutes, 51.54 seconds for her 12-and-a-half laps in the penultimate heat of eight alongside Ireen Wust of Netherlands, who took silver after finishing 2.74 seconds back.
The 26-year-old's three golds, one silver and one bronze putt her just ahead of her compatriot, triple Olympic javelin champion Jan Zelezny, who won two of his medals competing for the former Czechsolovakia.
"My greatest feeling right now is that my feet hurt, but I'm very happy," the shy Sablikova told reporters.
"I was really nervous, I couldn't sleep until 0200 last night, when I realised 5,000 people came to watch today, I got even more nervous.
"It's great to win it here in Sochi as well, and this time in front of all my family. They came here today especially for this race, and it's just great to win."
Sablikova was dubbed the "queen of skating" four years ago in Vancouver after winning the 3,000m and 5,000m Olympic titles but, like most at the Adler Arena, had already tasted defeat at the hands of the Dutch, who had won six of nine races on the oval.
Wust had pushed her into second place in the 3,000m on the opening weekend of competition in Sochi and started the longest women's race at a powerful pace.
Sablikova, meanwhile, had to rearrange her sunglasses on her opening laps as she fell 2.5 seconds behind her rival but she did not panic.
Wust is more proficient over the shorter distances and world record holder Sablikova reigned her in with three laps remaining. Once in front she never looked like giving up her advantage.
The 26-year-old then took a front row position by the finish line to watch the final pair, German Claudia Pechstein and Yvonne Nauta of Netherlands, who could not match the stamina and speed of the Czech.
Sablikova, who also won a bronze in the 1,500m in Vancouver, punched the air in delight upon confirmation of her victory and skated off to the corner of the Adler Arena to acknowledge a pocket of passionate Czech fans.
Carien Kleibeuker of Netherlands took bronze, the 21st Dutch speed skating medal out of 30 in Sochi, with home favourite Olga Graf just 0.11 seconds behind in fourth.
Wust said she was pleased with silver, her third of the Sochi Games after second place finishes in the 1,000m and 1,500m.
"In the 5000m I only have a chance when I start fast, and I always die towards the end. I skated a personal best, so I have to be satisfied," Wust told reporters.
"She's really strong, and she's built for the long distances, she always does well. There is a lot of pressure on her to win and she is a great champion," she added of Sablikova.
The Olympic medal was Wust's seventh, leaving her two short of Claudia Pechstein's record of nine. The 41-year-old German was unable to add to her tally with a fifth place finish on Wednesday.
Pechstein said she had no plans to quit after finishing 6.85 seconds behind Sablikova.
"I delivered my best. It's too bad I didn't win a medal, but the competition was tight. It was a hard race. It's a victory for me to be here again," she said before expressing her delight at a rare Dutch failure.
"It's nice that the Dutch didn't go home with a gold medal."
- Sports & Recreation
- Ice Skating
- Claudia Pechstein