Abstract: Strains with antagonistic effect against Diaporthe longicolla which causes soybean Diaporthe stem blight were screened by plate confrontation method, etiolated hypocotyl inoculation and pot experiment from 158 endophytic fungi isolated from healthy soybean. The results showed that 7 strains which could inhibit the mycelia growth of D. longicolla on PDA and the infection of D. longicolla to soybean were identified, among which six strains were preliminarily identified as Fusarium species. Furthermore, the pot experiment verified that the strains SR62 and SR115 with stronger antagonistic effect could significantly improve the germination rate, fresh weight and plant height of soybean treated with D. longicolla, but could not directly promote the growth of soybean plants. SR62 and SR115 were identified based on morphological and phylogenetic analysis, which were clustered in a clade together with F. falciforme strain NRRL32721 and classified as F. solani species complex(FSSC) clade 3. Compared with SR62 and SR115, the six highly pathogenic Fusarium strains had no significant effect on the mycelial growth of D. longicolla. In conclusion, SR62 and SR115 with strong antagonistic effects were screened, which could be used to control soybean Diaporthe stem blight.