Pseudomonas fluorescens lipase (PFL) was covalently immobilized on carbon nanofiber (CNF) using 1‑ethyl‑3‑[3‑dimethylaminopropyl] carbodiimide (EDC)/N‑hydroxysuccinimide (NHS). Surface functionalization of carbon nanofiber augments dispersibility as well as efficiency of covalent immobilization. Crucial parameters for immobilization such as pH, enzyme-support ratio, reaction time and mixing rate were optimized using one factor at a time (OFAT) approach. The nanobiocatalyst prepared under optimized conditions demonstrated a ten-fold increase in enzyme activity and the advantage of high thermal stability (up to 85 °C) along with 10 cycles of reusability. Subsequently practical application of the nanobiocatalyst was explored in the kinetic resolution of racemic 1‑phenylethanol into (S)‑1‑phenylethanol [C = 49.1%, eep = 99.5%, ees = 98.5% and E value = 151.4] followed by Mitsunobu reaction with a substituted pyrrole, giving an enantiopure (R)-carboetomidate analogue (yield = 83%).