Abstract: In order to clone and express a keratinase gene from a Bacillus strain,13 keratinase genes form Bacillus was classified to do multiple sequence alignments. The keratinase gene was cloned and expressed successfully in Escherichia coli by using degenerate primers. Finally,the expression of recombinant enzyme was confirmed by SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)and enzyme activity assay. Simultaneously,the results of temperature optimization experiment showed that the best induction temperature of the recombinant strain E. coli BL21(pET-28a-ker)was 20 ℃,and the activity of crude enzyme solution reached 389.7 U/mL after induction of 0.5 mmol/L IPTG for 16 h. This paper demonstrates the effectiveness of degenerate primers for keratinase designed by multiple sequence alignments,and this method can simplify the research and development of keratinase.