Abstract: Pseudomonas aeruginosa can effectively antagonize Arthrinium phaeospermum (Corda)M.B.Elli. Single-factor experiment and orthogonal tests were used to optimize the fermentation medium and culture conditions of Pseudomonas aeruginosa, and the characters of its antibiotic substance were also investigated. According to orthogonal analysis, the optimal formula of fermentation medium was sucrose 5.0 g, peptone 10 g, NaCl 2.5 g, and distilled water 1 000 mL. The optimal fermentation conditions were determined as follows: fermentation for 60 h at 25 ℃ with an initial pH at 7.5, an inoculum size of 1 mL and 150 mL medium volume in 300 mL triangle flask bulk. The molecular tests showed that the molecular weight of its active ingredient was less than 1 000 u; the biological activity of macromolecular proteins in its metabolites was far lower than that of the low-molecular-mass non-proteins; most active constituents in the crude extracts were small polarity substances, which had better solubility in organic phase, and the best extracting agent of which was methenyl chloride.