Abstract: The crude enzyme solution of cortical cortex-lytic was purified by ammonium sulfate precipitation,anion exchange chromatography and gel filtration. The results showed that when the saturation of ammonium sulfate was 60%,most of the cortical cortex-lytic enzyme was accumulated and precipitated,and the total protein and cortex-lytic enzyme activity were higher. The total protein and the activity of cortical cortex-lytic enzyme were higher,the specific activity of the enzyme was 158.22 U/mg,and the recovery rate was 84.17%. After further purification by Sp-sephadex C-25 ion exchange chromatography,the specific activity of this enzyme was 218.31 U/mg,and the recovery rate was 68.43%. Finally,Superdex 75 gel filtration was used to purify the cortex-lytic enzyme with high purity and the total enzyme activity was 1690.75 U/mg,and the specific activity of the enzyme was 1690.75 U/mg,and the recovery rate was 19.45%. The purity was 14.98 times that of crude enzyme solution. The molecular weight of purified cortex-lytic enzyme was 61.1 kDa. Fourier transform infrared spectroscopy(FTIR)was used to analyze the enzyme structure of Bacillus subtilis,and the absorption peaks at 3415,1665,1080,528 cm^-1 wavenumbers,and there were obvious amide I bands at 1665 cm^-1. The secondary structure of Bacillus subtilis cortex-lytic enzyme was studied by second derivative and curve fitting. It was found that there were 12.80% α-helix,31.56% β-sheet,44.97% β-turn and 10.68% random coil in the enzyme. This paper provided a cortex-lytic enzyme for studying the mechanism of HPTS killing spores.