Mixed culture of Trichoderma reesei and Aspergillus niger was employed to accomplish on-site cellulase production where cellulases were applied directly to the enzymatic hydrolysis of pretreated corn stover. We comprehensively compared the five pretreatments including sodium hydroxide, steam explosion, aqueous ammonia, lime and diluted sulfuric acid in the enzymatic hydrolysis and then in the whole processes from corn stover to single cell oil (SCO). The results were not completely but roughly the same. However, it is conclusive that sodium hydroxide pretreatment was the best one. The process with sodium hydroxide pretreatment could produce 23.5 g dry cell biomass harboring 13.7 g single cell oil (SCO) from 146.2 g corn stover. The total yields of cell biomass and SCO were 0.161 and 0.094 g g⁻¹ corn stover, respectively. This process was proven the most efficient. Through this work, we established efficient SCO production process from corn stover.