Abstract: In this paper, using lipase activity as index, fermentation liquid medium composition and culture conditions for lipase production by Neurospora sitophila were determined by single factor and orthogonal experiment. The results showed that the optimum conditions were as follows: the medium was composed of wheat bran and soybean meal, and the ratio of wheat bran and soybean meal was 5∶ 3, 1.0% of tween 80 and 0.1 g/L of KCl were added to the medium, initial p H of the medium was 6.0, liquid medium volume was 50 m L in 250 m L triangle bottled, inoculation quantity was 1%, rotate speed was 150 r/min, culture time was 72 h and culture temperature was 28 ℃. Under the optimum conditions, lipase activity reached 377.51 U/m L which was improved by about 6 times than that before optimization. The lipase production from Neurospora sitophila was enhanced effectively by optimization.