The present study reports production, partial purification, and media optimization for alkaline protease using Bacillus cereus PW3A. A profiling study for protease production indicates maximum enzyme activity (17.22 U/ml) was observed after 48 h of incubation. The studies also showed that the enzyme activity increased with the decrease in carbon content indicating the growth associated with nature protease production. Partial purification of protease was done using ammonium sulfate precipitation and dialysis. Further studies were conducted to assess significant media ingredients influencing protease production using the one-factor-at-a-time approach and Plackett-Burman design. Fructose and yeast extract were identified as the most significant variables. Response surface methodology was applied to optimize the factors for maximizing protease production. The results showed that the production increased from 17.22 U/ml to 47.43 U/ml indicating a threefold augment in enzyme activity. Characterization of protease showed that the highest enzyme activity was shown at pH 8.0 and temperature 50°C; however, significant enzyme activity was retained till pH 10 and temperature 60°C. Using casein as substrate, the enzyme showed maximum activity V max 39 U/ml and K m 18 μM. The activity was enhanced by MgCl 2 and CuSO 4 and inhibited by HgCl 2. Since the enzyme has both pH and temperature stability with greater substrate affinity, this protease finds many useful industrial applications.