The nature of immobilised Candida antarctica lipase-B (CAL-B) catalysed hydrolysis of tuna fish oil was studied with parameters such as solvent and water concentration, temperature, speed of agitation and enzyme loading. Immobilised CAL-B and support material immobead-150 were characterised with BET surface area, particle size analyser and Fourier transform infrared spectroscopy (FT-IR) to record their physiochemical properties. The maximum rate of reaction (Vmax) of 500 µmol of free fatty acids (FFAs) per mL reaction mixture per h and Michaelis−Menten constant (KM) of 2115 µmol FFAs/mL were found for Michaelis−Menten type kinetic model. Activation energy (E) of 26.1 KJ/mol was calculated for immobilised CAL-B. The 55.9% conversion of triglycerides was observed after the third use of the immobilised CAL-B. The activity retention of immobilised CAL-B reduced to 33.5% after the fourth repeated use of the enzyme.