Highly sensitive electrochemical lactate biosensor was constructed by one-step preparation route of amperometric enzyme electrode based on carbon nanotubes (CNT) and biocatalyst within an electropolymerized polypyrrole (PPy) film electrodeposited on stainless steel electrode. The nanobiocomposite film was electrochemically synthesized by electro-oxidation of pyrrole in a neutral pH solution containing appropriate amounts of lactate oxidase (LOD) and functionalized multi-walled carbon nanotubes (c-MWCNT). The nanobiocomposite film was characterized by FTIR, SEM and EIS. The incorporation of CNT facilitated high enzyme loading, increase in lifetime, stability and fast response time of the enzyme electrode. In present work artificial sweat was used as a non-invasive analyte for detection of lactate. When PPy/c-MWCNT/LOD modified electrode tested for lactate in artificial sweat, the apparent Michaelis–Menten constant (Ḱm) and maximum current (Imax) were found to be 0.833mM and 2.5μA respectively. The sensitivity and the detection limit were found to be 0.0778μA/μM and 5.61μM/l respectively with a linear response range from 5 to 60μM (R2 =0.95). The response time and shelf-life were found to be 8sec and 2 weeks, indicating good sensing results.