Keratinase, a vital enzyme in hair degradation, requires enhanced stability for industrial applications in the harsh reaction environment used for keratin hydrolysis. Previous studies have focused on improving keratinase thermostability. In this study, directed evolution was applied to enhance the organic solvent stability of the keratinase BLk from Bacillus licheniformis. Three mutants were identified, exhibiting significant enhanced stability in various solvents, although no similar improvements were observed in terms of thermostability. The identified mutations were located on the enzyme surface. The half-lives of the D41A, A24E, and A24Q mutants increased by 47-, 63-, and 61-fold, respectively, in the presence of 50% (v/v) acetonitrile compared to that of the wild type (WT). Similarly, in the presence of 50% (v/v) acetone, the half-lives of these mutants increased by 22-, 27-, and 27-fold compared to that of the WT enzyme. Notably, the proteolytic activity of all the selected mutants was similar to that of the WT enzyme. Furthermore, molecular dynamics simulation was used to assess the possible reasons for enhanced solvent stability. These results suggest that heightened intramolecular interactions, such as hydrogen bonding and hydrophobic interactions, contribute to improved solvent tolerance. The mutants obtained in this study hold significant potential for industrial applications.