Abstract: In order to search for new biocontrol agents for tobacco root knot nematode, strain TB-68 was identified by morphology and 16S rDNA sequence analyses. Its control efficiency against tobacco root knot nematode was evaluated through in vitro activity tests in greenhouse and pot experiments. The results showed that strain TB-68 was identified as Bacillus velezensis. The relative mortality of the second stage juvenile of root knot nematode and the inhibition rate of egg hatch due to TB-68 fermentation filtrate reached 91.7% and 81.8%, respectively. The control efficiency of the treatment using 200 mL fermentation filtrate per plant in the greenhouse reached 64.4%. In addition, TB-68 fermentation filtrate could significantly reduce the contents of total sugar and total protein in the eggs of root knot nematode. The results indicated that strain TB-68 had a great potential for the green control of tobacco root knot nematode.