Abstract: Positively charged Bacillus subtilis neutral proteinase dissolved in the aqueous phase at a pH value below its pI could form hydrophobic ion-pair with the anionic surfactant AOT in isooctane when they are mixed together due to electrostatic attraction. Thus，the hydrophilic enzyme could be “solubilized” into the hydrophobic isooctane. In this paper，the factors affecting the ion pair formation were investigated，such as，mixing conditions of the two phases，initial pH value and ionic strength，salts，the concentrations of the enzyme and surfactant AOT. The results indicated that magnetic stirring at 120r/min for 6 h was better than other mixing methods. The initial pH value of the aqueous phase affected both formation of enzyme-surfactant ion pair and transfer of the enzyme into isooctane. The optimal pH value was about 5.0. Addition of CaCl2 could partially prevent emulsifying during mixing，but excessive CaCl2 could negatively affect formation of ion pair and transfer of the enzyme from aqueous phase into isooctane. The transferred amount of enzyme into isooctane increased when AOT concentration increased within the limit (below the critical concentration of reverse micelle formation，4.9 mmol/L)，although the initial concentration of the enzyme in the aqueous phase was kept constant. However，with a constant AOT in isooctane，the enzyme transfer increased with increasing initial concentration of the enzyme in the aqueous phase first but decreased with further increase. The results showed that Bacillus subtilis neutral proteinase extracted into the organic phase was still active and could catalyze the transesterification.