Abstract: Abstract: Through the dilution method, 13 antagonistic endophytic bacterium were isolated from the infected tabacoo laterals. Among these antagonistic bacteria HN3 was exhibited the highest antagonistic activity against Ralstonia solanacarum. HN3 has been identified to be Pseudomonas with BIOLOG automatic microbiology analysis system and 16S rDNA determination and analysis. For the high production of antimicrobial substance growth. the medium composition were optimized by single factor carbon﹑nitrogen﹑inorganic and orthogonal experiments L9(34). As a result, The optium medium were glucose 12g、carbamide 0.32g and potassium phosphate 3g. after the optimization, antagonistic activity of the strain HN3 enhances 35.7%.