Abstract: The aim of this study was to identify the type of siderophore secreted by Pseudomonas aeruginosa Gxun-2, determine the optimal fermentation conditions and investigate the antagonistic effect of fermentation broth on plant pathogens, so as to provide theoretical basis of the strain in agriculture. The chrome azurol sulfonate (CAS) plate and full-band ultraviolet light absorption method was to qualitatively and quantitatively analyze the siderophore production of P. aeruginosa Gxun-2. The iron-producing medium and culture conditions were optimized by the combined method of single-factor, Plackett-Burman and orthogonal tests. Finally, the plate confrontation method was used to assess the antagonistic effect of its fermentation brot on the pathogenic fungus, Fusarium oxysporum f. sp. cubense (FOC4). The results showed that the type of siderophore produced by strain Gxun-2 was isohydroxamic acid type, and the optimal fermentation medium and culture conditions were determined as follows: sodium succinate 2 g/L, glycerol 10 g/L, ammonium sulfate 1 g/L, initial pH6.8, temperature 35 ℃, inoculum 1% (v/v), and loading volume 140/250 mL. Under these conditions, the siderophore activity unit (Su) of the Gxun-2 fermentation broth reached 81.20%±0.24%, which was 82.84%±0.82% higher than that before optimization, and the inhibition rate on FOC4 reached 70.60%±1.87%, which was 52.68%±4.23% higher than that before optimization.