The production of sterol ester by transesterification of β-sitosterol with fish oil (TAG) catalyzed by Thermomyces lanuginosus immobilized lipase enzyme with varied reaction parameters such as temperature, substrate molar ratio, concentration of enzyme to deduce the enzyme kinetics for the reaction was investigated. For this transesterification reaction, the kinetic model was derived by using Ping Pong Bi Bi Mechanism. The Km value from the first plot containing fish oil as substrate was 1.31±0.05, while Km from the second plot containing β-sitosterol as substrate was 1.01±0.04; identical Vmax (0.213±0.06) values were obtained by keeping one of the substrate concentration constant and varying the other. Practical applications: Deduction of reaction kinetics is an important criterion to ascertain the viability of any chemical process. Enzymatic processes need special attention to set model reaction parameters which could help in optimization or design of the actual process. In the present study we have derived the enzyme kinetics for the production sterol ester, an important nutraceutical, and calculated its Km and Vmax values along with the Arhenius activation energy to establish the viability of the reaction. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.