Smyth, 30, who is visually impaired, had already won 100m gold and remains undefeated in both events in every Paralympics and Worlds since 2008.
Great Britain failed to win a medal on day five of the championships - having won gold on every day so far.
GB's T42 long jumper Luke Sinnott came fourth in his debut at a major event.
And there was a fourth-place finish for team-mate Richard Chiassaro in the T54 200m, with compatriot Nathan Maguire sixth.
Elsewhere for Britain, Sabrina Fortune, who won F20 shot put bronze at Rio 2016, could only come sixth, while teenager Polly Maton was fifth in the T46/47 100m.
In the morning session, Sammi Kinghorn came second in her heat to progress to Wednesday's T53 400m final, where she will look to add to the 200m title she won on Saturday.
GB are third in the medal table with 11 golds and 20 total, with the US leading and China second.
Smyth, born in Northern Ireland, holds the world record in the T13 100m and 200m - his times of 10.46 and 21.05 seconds are quicker than the records from any other classification.
A legally blind athlete, he has won 10 Paralympic and world titles and has raced in able-bodied events at the European Championships.
"This is incredible. Today you actually get to enjoy it. In the 100m on Sunday I knew I was back out again and had to rein it in," he told BBC Radio 5 live.
"I'm delighted to be back in London. I wish we could have the Worlds in London every time."
Sinnott, 36, lost both of his legs in an improvised explosive device (IED) explosion while serving with the Royal Engineers in Afghanistan in 2011.
He has gone to compete in sailing and has raced at the Invictus Games, before committing to long jump last year.
The Hampshire athlete's best jump of 6.15m came after four fouls from his first five attempts and was 10cm short of third-placed German Leon Schafer, with Atsushi Yamamoto of Japan taking silver and Denmark's Daniel Wagner winning with a jump of 6.50m.
"I really had to go into a deep, dark place to get what I needed for that last jump. I knew if I didn't put something on the board in that last jump then I'd be very disappointed," he said.
"I can't believe I'm here. Those days you wake up and feel fate dealt you a bad hand, it just goes to show that if you take what's handed to you and you turn it around then you can become part of amazing stuff like this.
"There's a sensitive tipping point where you either end up here or you end up feeling sorry for yourself. Strapping a pair of blades was one of the best things I ever did."
Maton, 17, was a spectator at London 2012 and has been earmarked as a prospect for the Tokyo 2020 Paralympics.
"I'm quite happy, the time wasn't great but I've had calf problems the last couple of days. You just try to be in the moment and forget about it," she said.
"It's a complete dream to be here - I was here at London 2012 and I came back for the grand prix in later years. It's so great to be in the stadium and running."
Chiassaro, competing in his third of four events at these championships, recorded a season's best of 25.12 seconds, while Maguire put in a personal best of 25.86 seconds. Both race in the 400m on Thursday.
Canada's Brent Lakatos claimed his second gold of the championships, adding the T53 400m to his 200m title and the 800m still to come.
The 37-year-old is now a nine-time world champion.
"I have a little headache and can't breathe through my nose but I gave it everything. It feels amazing to win this," he said. "I went to Rio full of confidence but didn't win. l got my butt kicked a couple of times."
American Tatyana McFadden moved a step closer to completing a T54 quadruple by progressing through her 800m heat. The seven-time Paralympic gold medallist has already claimed the 200m, 400m and 1500m titles in London.
Ireland's Jason Smyth won T13 200m gold to complete a sprint double at the World Para-athletics Championships in London.