The sisters took advantage of a nervy start from their opponents by breaking in the opening game of the match.
The Americans looked comfortable on serve for the majority of the set but were almost broken back when attempting to serve it out at 5-4.
Hlavackova and Hradecka saved two set points at 40-15 before earning a break point of their own but they couldn't convert and Venus produced some first serves to eventually clinch the set.
The next service game of the sisters was also a tight affair, with the Czechs squandering two chances to move ahead an early break in the second.
They were soon punished for this as the Williamses took 12 points in a row by holding twice to-love and also breaking to-love to take a 4-2 lead.
The Czech duo stopped the run of games in the next but soon it was down to Serena to serve out her second gold medal of the Games.
She moved to 40-15 and two gold-medal points but the Czechs wouldn't lie down, saving both to bring it back to deuce.
However, Serena found an ace to set up gold-medal point number three and this time Venus put away an easy volley to seal gold.
The sisters become the first tennis players to win gold at three separate Olympic Games.