Fungal strains LX6F2 and YGF9 were respectively isolated from poplar soil and poplar tissues, both of which have strongly antifungal activity to pathogenic Botryosphaeria dothidia of poplar canker. In order to improve the antifungal activity of the strains, the fermentation technology was optimized by response surface method. The optimum carbon source, nitrogen source and inorganic salts were determined by single factor experiments. The suitable concentration of carbon, nitrogen source and the basic fermentation conditions were found by orthogonal experiments. The results showed that the effects of the 8 factors on the antifungal activity of LX6F2 strain were not significant by Plackett-Burman design, so the optimized fermentation medium and conditions for strain LX6F2 were as follows: potato juice 200 g / 1000 mL, fructose 1%, beef powder 1.4%, KCl 0.1 %, initial pH value 7.0, the temperature 32°C, shaker speed 180 r/min, inoculation amount 4%, fluid volume 100 mL / 250 mL. The fermentation conditions for strain YGF9 was optimized by central composite design and response surface analysis. Through response surface plot and contour plot analysis, the optimized fermentation medium and conditions for strain YGF9 were as follows: potato juice 200 g / 1000 mL, fructose 1%, beef powder 1.4%, KCl0.1 %, initial pH value 7.0, the temperature 34.14°C, shaker speed is 188.80 r/min, inoculation amount 3.58%, fluid volume 100 mL / 250 mL. The inhibition rate of the strains could reach 75.88% and 84.98% respectively. These results improved the antifungal activity of the two biocontrol strains, and also provided guarantee for exploitation and utilization of biological agents to bio-control of poplar canker.