Abstract: Objective:Organic-solvent tolerant protease-producing strains were isolated from polluted area in Yanwei Port,the taxonomic status were clarified,and the enzymatic characteristics of the protease were studied.Methods:Organic-solvent-tolerant strains were isolated from organic-pollution water by adding 20%(V/V)methylbenzene into medium as selective pressure. Protease-producing strains was obtained through the first screening with selection medium plate. The high-producing strains were re-screened by shake flask fermentation. Then the strains were identified and the enzymatic characterization was measured. Results:MSP05 was obtained and identified as Bacillus amyloliquefaciens by its morphological,physiological,biochemical characteristics and 16S rDNA sequence homology. The optimum pH and temperature values of this protease were pH10 and 50℃,respectively. The enzyme was activated by Cu²⁺,Mn²⁺ and inhibited by Mg²⁺,Fe³⁺ and Ba²⁺. This protease has good organic-solvent tolerant. It remained more than 50% activity after 72 h incnbated with 50% enthol at 25℃ and 140 r/min.Conclusion:One high-producing organic-solvent tolerant protease producer Bacillus amyloliquefaciens MSP05 was obtained,which would have potential industrial value.