Subcritical co-solvents of n-hexane/isopropanol were primarily utilized to extract lipid from wet microalgal pastes of Scenedesmus obliquus. The effects of key operational parameters were investigated, and the optimal parameters were obtained: solvent ratio of n-hexane to isopropanol was 3:2 (V:V), phase ratio of co-solvents to microalgal biomass was 35:1 (mL:g), reactor stirring speed was 900 rpm, extraction time was 60 min. Additional pretreatment with acid, ultrasonic and microwave as well as enhanced subcritical pressure/heating treatments were also applied to further study their effects on lipid extraction. The results showed that the lipid recovery rate with acid pretreatment was 8.6 and 6.2% higher than ultrasonic and microwave pretreatment; the optimum enhanced subcritical condition was 55 °C with atmospheric pressure. Under optimal operating conditions, the lipid and FAME yield were 13.5 and 7.2%, which was 82.6 and 135.1% higher than the traditional method. The results indicated that the subcritical n-hexane/isopropanol extraction process had promising application potential.