The six-time Olympic champion will race for the seventh time at the meet, which is part of the new IAAF World Challenge series. It is scheduled for June 27.
Bolt will join Jamaican teammate Yohan Blake, the 2011 world champion in the 100 metres, for the 4x100-meter relay.
Blake is expected to be Bolt's toughest challenger at the Aug. 10-18 world championships in Moscow. The other two names on the relay team have yet to be named.
"I'm looking forward to running the 4x100m in Ostrava this year," Bolt said. "The meeting is only a few days after the Jamaican Championships and the relay fits nicely into my competition program."
Bolt is so far committed to run in Europe this year in the 200 metres at the Bislett Games in Oslo, Norway, on June 13.
Bolt won the 100 metres in Ostrava last year in 10.04 seconds before he defended his titles in the 100 and 200 at the London Olympics and anchored the Jamaicans to victory in the 4x100 relay in 36.84 seconds to set a world record.