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Proteases are the type of hydrolytic enzymes which have potential biotechnological applications. We report the isolation, optimized production, purification, and cell culture applications for halo-tolerant protease from Salinicola tamaricis BGN2, isolated from the marine environment on the West coast of India. Media optimization for protease production was done using a central composite design by response surface methodology. The initial screening and Plackett–Burman design revealed that glucose, fish glue, soybean flour, CaCl2, and NaCl were significant factors for protease production. A fivefold increase (55.2 U/ml) in protease production was observed with an optimized medium comprised of glucose (0.5%), fish glue (0.75%), and NaCl (10%) after 96 hours of incubation. Acetone precipitation and gel-filtration chromatography followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and zymography analysis showed a 43 KDa molecular weight for the purified protease. Application studies demonstrated significant trypsin-like activity of protease in sub-culturing MCF7 cell-line in both time and dose-dependent manner. Effective time and quantity of protease for cell dislodging activity were found to be one minute and 200 μl, respectively. It is a rare report demonstrating the trypsin-like activity of proteases for cell culture applications.