A strain with high keratinase activity of 46.89 U(mL^-1 was isolated from poultry plant soil samples by using feather powder as the sole carbon source and nitrogen source. The strain was identified as Bacillus subtilis KS12 by the morphological characterize, and 16S rRNA sequence analysis. The effects of pH, temperature, ions, surfactants, oxidants and organic solvents on the keratinase activity of B. subtilis KS12 were investigated. The result showed that the optimum pH and temperature of the keratinase of the strain were 7.5 and 37℃, respectively. The keratinase was stabile between pH 8.0 and 9.0 with the relative enzyme activity of 95.5%~97.2% for 1 h, and the relative enzyme activity were 61.4% and 59.5%, respectively, at 60 ℃ and 80 ℃ for 1 h. The keratinase activity was improved by 5 mmol(L^-1 Ca²⁺ and Mg²⁺, especially Mg²⁺ with the relative enzyme activity of 159%, and inhibited by Cu²⁺ (5 mmol(L^-1) with the relative enzyme activity of 3.7%, and 5% of Tween 80 expressed a positive effectt on the keratinase activity with 140.4%. Furthermore, the keratinase activity of B. subtilis KS12 were inhibited by isopropanol, formaldehyde, methanol, ethanol, isoamyl alcohol and xylene.