The Williams sisters, who also won doubles gold in Beijing and Sydney, took an hour and a half to wrap up their defeat of the number four seeds under the Centre Court roof.
"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," said Serena, who also won the women's singles gold on Saturday.
The Czech pair saved two match points before Venus secured the victory with a backhand volley, hugging her sister in celebration as chants of "USA! USA!" rang out from the crowd.
With Venus also having won singles gold in Sydney, the pair have overtaken previous record holder Reginald Doherty of Britain, who won a total of three golds in 1900 and 1908.
"It's incredible. After 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. Wow. It just feels unreal," said Venus, who is already thinking about adding a fourth doubles gold to the collection at the 2016 Games.
"We're definitely planning on Rio. We're going out with a bang in Rio. Hopefully we will make it four, you never know."