In the present study, the production of keratinase from Stenotrophomonas maltophilia strain Kb2 was optimised. Plackett-Burman Design (PBD) followed by Central Composite Design (CCD) were used for the determination of significant variables for keratinase synthesis, and the concentration of three variables—feather meal (FM), starch, and MgSO4—were optimised for higher keratinase production. Further an interaction of the selected variables, Partial Least Square Regression (PSLR) analysis was performed. Optimised culture medium led to maximum keratinase activity of 70.8 ± 3.9 U/ml after 24 h of growth, which was 4.91 fold higher than the keratinase obtained from un-optimised media. The keratinase yield was very close to the real projected value. The statistical and multivariate analysis assisted in maximising the keratinase production. Further, crude keratinase was studied for its utilization as a dehairing agent. The results of the present study clearly showed enhanced production of keratinase by employing PBD and CCD followed by the PSLR approach.