Jamaica’s Bolt has already won the 100m and is now aiming for an eighth Olympic gold by retaining his 200m title.
But American Gatlin, who won silver behind Bolt in the 100m final, only finished third in his semi-final.
Britain’s Adam Gemili, 22, reached the final as one of two fastest losers.
Gatlin, 34, banned twice for doping offences, clocked 20.13 and failed to progress along with another big name – Yohan Blake.
The Jamaican, who won silver behind Bolt in London four years ago, has been hampered by injury over the past couple of years and faded to finish sixth in 20.37 seconds.
LaShawn Merritt, who has run 19.74 this year, is likely to pose the biggest challenge to Bolt in the final.
But Bolt believes he can break his world record of 19.19 and could even go under 19 seconds in the final.
“I definitely think I can try for the world record, I definitely feel that,” he said. “I need to run efficiently and get into the straight and run the perfect race.”
Bolt said he would have a better chance of achieving his goal to run under 19 seconds if he gets a high lane, adding: “If I get a lower lane it’s always harder for me to run the corner when it is tighter.”
On Gatlin’s exit, Bolt told BBC Radio 5 live: “Everybody’s in shock. You can tell from the 100m he’s getting old. It’s a fact the older you get, the rougher it gets to double.
“Next year if I go to the World Championships [in London] I’m not going to double. I’m going to do the 100m and that is it.”
Bolt has won all seven of his previous Olympic finals, claiming gold in the 100m, 200m and 4x100m relay at both Beijing 2008 and London 2012.-BBC