Serratiopeptidase is a proteolytic enzyme obtained from serratia marcescens strain E-15 and used as anti-inflammatory and analgesic drug. Serratiopeptidase undergoes first pass metabolism, causes the gastrointestinal disturbance and systemic toxicity after oral administration. To overcome the limitations of serratiopeptidase, transdermal drug delivery system is an alternative method. So, the aim of present work was to modulate serratiopeptidase transdermal patch by lipid-based transfersomes. Particle size of drug was the major concern to cross stratum corneum which acts as a barrier. This difficulty was surmounted by modulating the vesicles such as transfersomes which carries the drug into the skin by passing the barrier of stratum corneum. Serratiopeptidase was encapsulated in transfersomes in different ratios of lecithin and cholesterol. Particle size of transfersomes, folding endurance, thickness, tensile strength, adhesion test, encapsulation efficiency, in vitro and in vivo release, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimeter (DSC) studies of patch were used as characterization parameters. Serratiopeptidase transfersomes size was found to be 50 mu m with smooth surface. The promising entrapment efficiencies of transfersomes and formulation were found to be 96.76 and 98.7%, respectively. In vitro and in vivo release studies showed controlled and steady release of serratiopeptidase for 24h. FTIR and DSC confirmed the encapsulation of drug in patch without interaction. It is concluded that transfersomes are interesting carriers for enzymatic drugs for topical application.