In this article, the optimum culture medium formula and the optimal culture conditions for Pleurotus citrinopileatus fermentation were determined through culture of Pleurotus citrinopileatus Sing using single factor experiment with a combination of orthogonal design and response surface method. The results showed the optimum culture medium formula were glucose 1.00 %, sucrose 1.00 %, peptone 0.3 %, potassium dihydrogen phosphate(kdp) 0.15 %, NaCl 0.03 %, magnesium sulfate heptahydrate 0.07 % and Vitamin B1 0.001 %; the optimal cultivation condition was as follows. Inoculation amount, 5 %; 150 mL liquid medium inoculated into a 250 mL shake flask; cultural temperature, 27 ℃; initial pH value , 6.55; rotary speed , 155 r·min^-1.