The current work was attempted to isolate and characterize the serratiopeptidase producing Serratia sp. Among the 10 bacterial isolates 7 strains were identified as Serratia sp. Out of 7 strains one showed potent proteolytic activity and selected for further studies. Based on the morphological, biochemical and molecular characterization, the potent isolate (RH03) was identified as Serratia marcescens (GenBank accession number: KC961637) and the strain was designated as Serratia marcescens VITSD2. The production of serratiopeptidase was carried out in trypticase soya broth and the enzyme was partially purified using ammonium sulfate precipitation and dialysis. The specific activity was determined by casein hydrolysis assay and was found to be 12.00, 21.33, and 25.40 units/mg for crude, precipitated and dialysed samples. The molecular weight of the protease was determined by SDS-PAGE and it was found to be 50 kDa. The antibacterial activity of the produced serratiopeptidase showed moderate activity against Pseudomonas aeruginosa MTCC No. 4676 (12 mm) and Escherichia coli MTCC No. 1588 (15 mm). © 2013 Higher Education Press and Springer-Verlag Berlin Heidelberg.