Abstract: Biodiesel is a fuel additive which has received considerable attention in the recent years due to their biodegradability and non-polluting ability. In the present study, an enzymatic route for biodiesel production from waste cooking oil was performed using methyl acetate as an acyl acceptor and immobilized lipase as biocatalyst. Microbial lipase from Candida rugosa was immobilized by entrapment method and was used for the transesterification of waste cooking oil into biodiesel. A good conversion yield was standardized by optimizing various parameters for the transesterification reaction. The produced biodiesel was subjected to Thin Layer Chromatography for the preliminary confirmation and was then confirmed using GC-MS. The fuel characteristics of the produced biodiesel were compared to that of the standards. These results prove that the biodiesel produced from waste cooking oil can be used as an alternate fuel in engines. From the results, the enzymatic transesterification of waste cooking oil into biodiesel via immobilized lipase has been evidenced as a better approach for the biodiesel production as it produces the product with high purity. The present approach was found to be effective and eco-friendly with environmentally benign novel technique.