In this study, we report the biochemical characterization of a novel serine protease from seeds of Cucumis maderaspatensis, aimed with assessing the anticoagulant and antiplatelet activities. The purified serine protease was obtained by subjecting the seed extract to ammonium sulphate precipitation followed by anion exchange and gel filtration chromatography. Twenty seven-fold purification with the specific activity of 884.2 U/mg of protease activity was obtained. The characterization of the novel protease enzyme activity for optimum temperature, pH and effect of different protease inhibitors and metal ions were measured using caseinolytic assay and casein zymogram. The relative molecular mass of the novel neutral serine protease (CmSP) is ~ 32 kDa. Its anticoagulant was determined by assessing the delay in plasma re-calcification time in both platelet-rich and platelet-poor plasma. The antiplatelet activity of serine protease was demonstrated by inhibition of agonists induced platelet aggregation; it was in the order of Epinephrine > Adenosine tri phosphate. Further studies would decipher the mechanism of action to understand its therapeutic potential as an antiplatelet and anticoagulant molecule.