Neutral protease from Bacillus subtilis was chemically modified by cyanuric chloride activated single methoxy polyethylene glycol( mPEG5000),then the enzymatic properties and the stability in the non-aqueous phase system were studied. Results showed that optimal temperature of the modified neutral protease by mPEG5000 was 50℃,which was in agreement with that of free enzyme. Furthermore,the relative activity was proportional to modification rate under certain pH. In addition,the modified enzyme exhibited more affinity with substrate,thermal stability as well as tolerance to a series of organic solvent compared with free enzyme. Our findings would be convenient for bio-organic catalysis in biphase or unique solvents.