Transesterification of linseed oil and pork lard mixture with ethanol was studied by applying a biotechnological method. The effectiveness of 16 lipases was evaluated, and the most effective lipase, Lipolase 100 L (EX), was selected for reaction condition optimisation by response surface methodology based on a Box-Behnken design. The influence of the main process parameters was evaluated, including the reaction temperature, ethanol to triglyceride molar ratio, lipase amount, and reaction time on transesterification yield. The optimal reaction conditions were found as follows: reaction temperature of 32.79 °C, ethanol to oil molar ratio of 5.99, lipase concentration of 6.39%, and reaction time of 7.95 h, with an expected transesterification yield of 52.2%. Optimised enzymatic pork fat and linseed oil mixture transesterification with ethanol was less effective compared with the chemical transesterifications described elsewhere; therefore, the step-wise transesterification procedure should be applied by adding ethanol and percent preparation in the reaction mixture after glycerol phase separation.