Seven kinds of alkyl alcohol (n-butanol, n-pentanol, n-hexanol, n-heptanol, n-octanol, n-nonanol, and n-decanol) were chosen as the cosolvent for Aliquat 336/ isooctane reversed micelles. By performing liquid-liquid extraction with Aliquat 336 reversed micelles as the extractant, the separation and purification of two enzymes (alpha-amylase and neutral protease) from Bacillus subtilis were investigated. Experiments revealed that when n-butanol is used as the cosolvent, the two enzymes from Bacillus subtilis can be effectively separated and purified after a full forward and backward extraction cycle. alpha-Amylase was separated in the stripping solution with about 85% of its total activity recovered and purified about 1.6-fold. Neutral protease was separated in the raffinate with about 80% of its total activity recovered and purified about 3.5-fold. For the other six alcohols used as the cosolvent for Aliquat 336/isooctane reversed micelles, separation was not achieved.