Extracellular protease production by Bacillus cereus isolated from the intestine of fish Mugil cephalus has been investigated in shake-flask experiment using different preparations of tuna-processing waste such as raw fish meat, defatted fish meat, alkali hydrolysate, and acid hydrolysate as nitrogen source. Among the tuna preparations tested, defatted fish meat supported the maximum protease production (134.57±0.47 U ml −1 ), and 3% concentration of the same was found to be optimum for maximizing the protease production (178.50±0.28 U ml −1 ). Effect of carbon sources on protease production in the optimized concentration of defatted tuna fish meat revealed that galactose aided the higher protease production (259.83± 0.04 U ml -1 ) than the other tested carbon sources and a concentration of 1.5% galactose registered as optimum to enhance the protease production (289.40±0.16 U ml −1 ). The halotolerancy of B. cereus for protease production indicated that 3% of sodium chloride was optimum to yield maximum protease (301.63±0.20 U ml −1 ). Among the surfactants tested, protease production was high in Triton X 100-added medium (298.63±0.12 U ml −1 ) when compared to other surfactants, and its optimum concentration recorded was 0.8% (320.57 ± 0.17 U ml −1 ) for more protease production. Partial characterization of crude enzyme revealed that pH 7.0 (278.90±0.08 U ml −1 ) and 60°C temperature (332.37±0.18 U ml −1 ) were optimum for better protease activity by B. cereus.