PEGylation is considered a successful technique to enhance the therapeutic and biotechnological potentials of peptides, proteins, toxins and drugs. The conjugation of polyethylene glycol (PEG) increases the size and molecular weight of conjugated molecule and improves its pharmacokinetics and pharmacodinamics by increasing water solubility, protecting from enzymatic degradation, reducing renal clearance and limiting immunogenic and antigenic reactions. These features are very useful for therapeutic proteins, since PEGylated proteins exhibit high stability and very low immunogenicity, ensuring a sustained clinical response with minimal dose and less frequent administration. The modification of snake venom toxins by PEGylation is a promising strategy to increase the use of these biomolecules in clinical practice, which has been limited by side effects of immune reactions in patients. Thrombin-like serine protease from Crotalus durissus collilineatus (SPCdc) is able to convert fibrinogen into fibrin and presents potential therapeutic application in cases of myocardial infarction, ischemic stroke and other thrombotic and vascular disorders. In this study we modified the SPCdc by site-specific PEGylation, producing the unique conjugate of molecular mass around 35 kDa, named SPCdc-PEG. Unexpectedly, the Km of the PEGylated enzyme (Km = 0.447 mM ± 0.025) was smaller than that of the native enzyme (Km = 0.770 mM ± 0.020), indicating that PEG-SPCdc has a higher affinity for the substrate TAME than SPCdc. Additionally, the values of Kcat/Km (1163 mM.min-1, for SPCdc-PEG and 350 mM.min-1, for SPCdc) showed that PEGylated enzyme has higher catalytic efficiency than the native form. These results demonstrated the relevant biopharmaceutical potential of SPCdc-PEG.