Abstract: Objective:The strain of SPE009 from Ginkgo biloba with high antibacterial property was identified and the fermentation conditions were optimized. Methods : By using morphological and molecular biological identification,the strain SPE009 was identified. Based on the single factor and orthogonal experiments,fluid volume,inoculum concentration,culture temperature,initial p H,carbon and nitrogen source were optimized with the inhibitory zone diameter of the broth. Results:The SPE009 train was identified Penicillium oxalicum. The formula of appropriated culture conditions were as follows:Glucose levels of 20g/L,liquid volume 30 m L/100 m L,p H 7.0,inoculum concentration 3%. Under these conditions,the resistance of Staphylococcus aureus antibacterial circle reached 17.6mm,improved by 21.4% compared with that under initial culture conditions. Conclusion:The metabolites of the endophytic fungi Penicillium oxalicum SPE009 from Ginkgo biloba had good antibacterial property,providing a new source of natural antibiont development.