Objective To optimize the medium composition and fermentation conditions of Penicillium citrinum PA-33 to improve its antibacterial activity. Method Single factor experiments were used to determine the optimum basic medium, carbon sources, nitrogen sources and inorganic salts. The optimum formulation of fermentation medium was determined by response surface methodology. On the basis of single factor tests of fermentation conditions, the optimal combination of fermentation conditions was optimized by ternary quadratic rotation unitized design and frequency analysis method. Result The optimal medium composition were: Potato juice 219.91 g·L^-1, mannitol 34.11 g·L^-1 and soybean powder 6.25 g·L^-1. The optimal fermentation conditions were: Liquid volume 50 mL, inoculation concentration 3.5% (φ), culture temperature 28 ℃, rotation speed 150 r·min^-1 and fermentation for 12 d. The inhibition zone diameter of fermentation broth after optimization on Escherichia coli reached 28.99 mm, increased by 10.26 mm compared with 18.73 mm under the original conditions. Conclusion Response surface methodology, ternary quadratic rotation unitized design and frequency analysis method significantly enhance the antibacterial activity of fermentation broth of Penicillium citrinum PA-33, and this study provides a basis for isolation of antibacterial active substances and industrial production of this strain.