Despite being short of their best form, they beat Andrea Hlavackova and Lucie Hradecka of the Czech Republic 6-4 6-4.
Serena, who won her fifth Wimbledon singles crown last month, adds doubles gold to her singles title.
The sisters first won the doubles at Sydney in 2000, then again in Beijing in 2008 after missing Athens because of an injury to Serena.
Venus believes they can go on to record a fourth victory at the Rio Games in 2016.
"It's incredible," she said. "Coming off Serena's victory, I think most of all we are happy to add to our country's medal count and be a part of it.
"We're definitely planning on Rio. We're going out with a bang in Rio. Hopefully we will make it four, you never know."
Serena, 30, becomes only the third female tennis player to win a pair of gold medals at the same Olympics, emulating Venus, 32, in 2000 and compatriot Helen Wills Moody in 1924.
The Czechs got off to a sluggish start when Hradecka's first service game was broken. The American duo, who withstood a break point as Venus served for the set, held on to take the opener.
The fourth-seeded Czechs made a more resilient start to the second set but Hlavackova was broken and the Williams sisters moved 3-2 ahead, never seemingly in any danger of letting their advantage slip.
"I would say this is more special than Wimbledon," said Venus. "I'm amazed she won the singles and our main goal was to win the doubles, so we got both and it's amazing."
Serena said that the doubles title was always her priority at the Olympics.
"It was so exciting to win the singles, but, like I said at the beginning of the tournament, my goal was to get gold in doubles here," she said.
"There's something about standing next to Venus and holding that gold medal. Three times we have played, three times we have got the gold medal. So we are pretty stoked about it."
Meanwhile Russian pair Maria Kirilenko and Nadia Petrova won the women's doubles bronze medal by beating top seeds Liezel Huber andLisa Raymond of the United States 4-6 6-4 6-1.