Serratiopeptidase is an extracellular enzyme produced by Serratia marcescens and possesses anti-inflammatory, analgesic, anti-atherosclerotic, anti-edemic, and thrombolytic properties. The objective of this study was to produce and characterize the serratiopeptidase from environmental isolates of Serratia marcescens. Serratia marcescens strains were isolated from soil samples by spread plate technique and identified by 16S rRNA-PCR assay. Trypticase soy broth was used as the production medium and the crude enzymes were prepared from the culture by centrifugation. The protein in the crude extract was measured by Lowry’s method. Further, the enzyme was purified by column chromatography, and the fractions were concentrated using ice-cold acetone. Serratiopeptidase in the fraction was confirmed by SDS PAGE and characterized for thrombolytic activity on human blood sample. 150 soil samples were processed and 21 isolates of Serratia marcescens were identified. The protein content in the crude enzyme preparation was estimated in the range of 9 to 12.4 mg/mL. SDS PAGE of the purified serratiopeptidase produced a distinct band with a molecular weight of 52 kDa. The significant thrombolytic activity produced by 15 isolates and the isolate SM24 shows the maximum thrombolytic activity of 95%. In this study, serratiopeptidase produced from the isolates of Serratia marcescens shows significant thrombolytic activity on the human blood clot. © 2023 Elsevier B.V., All rights reserved.