Abstract: In order to acquire Trichoderma strains with preferable inhibitory effect on tobacco black shank, twenty-eight strains of Trichoderma were isolated from rhizosphere soil of uninfected tobacco plants in Henan province. The strains of XYPQ-1, ZMD-1 and XYLS-5 exhibiting better inhibitory effects on Phytophthora nicotianae were screened out by plate confrontation, with inhibitory rate of more than 90% after five days. Based on molecular biological analysis, the three Trichoderma strains were identified as Trichoderma virens, Trichoderma koningiopsis and Trichoderma paraviridescens, respectively. The antifungal activity of metabolites and the antagonistic effect were also studied. The results showed that both the volatile and non-volatile metabolites of the three Trichoderma spp. had inhibitory effect on P. nicotianae, and the strain XYLS-5 exhibited the optimal inhibitory effect, with its inhibition rate of volatile and non-volatile metabolites achieving 61.71% and 100.00%, respectively. The three Trichoderma strains could control tobacco black shank in greenhouse, among which strain XYLS-5 performed the optimal control efficiency exceeding 80% and could promote seed germination and root growth of tobacco. Results indicated that strain XYLS-5 can be used as antagonist for controlling tobacco black shank.