The 25-year-old, who set a Games record in qualifying, clocked three minutes 31.615 seconds to beat Australian Annette Edmondson by 3.835 seconds.
Rowsell's compatriot Jason Kenny, 26, took silver in the men's sprint.
The three-time Olympic champion pushed New Zealand's Sam Webster all the way in the final, but went down 2-1.
"I am shattered," Kenny told BBC Sport. "I got a bit of cramp or something. I was throwing up a minute ago. I feel terrible."
Kenny started the event slowly on Thursday, qualifying 11th and losing his first-round match before reaching the last eight via the repechage.
However, he impressed in Friday's quarter-finals and semi-finals and, after losing the first race of the final, responded well to draw level at 1-1.
Glasgow 2014: Scottish duo take tandem time trialBut Webster proved stronger in the decider, beating Kenny on the line to take gold.
Earlier, Olympic team pursuit gold medallist Rowsell fell behind in her final, but quickly recovered to claim an advantage she did not relinquish.
"This is the one I really wanted," she told BBC Sport. "The Commonwealth Games come round every four years and very rarely near your home country.
"With the individual pursuit out of the Olympics now, this is the biggest event I can do in it.
"It's the event I was originally talent-spotted for when I started so it means a lot to me. I'm absolutely over the moon."
Scotland's Katie Archibald missed out on bronze in the same event as she lost to Australia's Amy Cure in the Sir Chris Hoy Velodrome.
England's double Olympic champion Laura Trott was edged into sixth during qualifying, while compatriot Dani King finished eighth, a place behind fellow Team GB world champion Elinor Barker of Wales.
Scottish Paralympic and world champion Neil Fachie, riding with pilot Craig Maclean, won the Para-cycling 1,000m tandem time trial for blind and visually impaired athletes.
The duo, riding last, beat Australians Kieran Modra and Jason Niblett by just 0.148secs with Matthew Ellis of Wales, piloted by Ieuan Williams, taking bronze.
Welsh endurance rider Owain Doull , 21, was beaten to bronze by New Zealand's Marc Ryan in the 4,000m individual pursuit.
Australian Jack Bobridge took gold from Alex Edmondson, who, like sister Annette, had to settle for silver.
Joanna Rowsell's gold was England's second in the cycling, after Sophie Thornhill and Helen Scott in the tandem B sprint 
Rowsell clocked a Commonwealth record time of 3:29.038 in qualifying 
Rowsell was diagnosed with alopecia aged 10 
Jason Kenny won race two of his final but could not add the Commonwealth Games sprint gold to his three Olympic titles 
New Zealand's Sam Webster took gold, beating Kenny 2-1 in the final
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