The abilities of three immobilized lipases from Candida Antarctica (Novozym 435), Thermomyces lanuginosus (Lipozyme TL-IM), and Rhizomucor miehei (Lipozyme RM-IM) to catalyze the transesterification of canola oil with methanol were investigated. Response surface methodology and central composite design were employed to optimization of the reaction parameters. Simplex lattice mixture design was employed to evaluate the effect of the composition of lipase mixtures on the yield of transesterification reaction. The highest conversion of canola oil to fatty acid methyl ester was obtained as 97.2%. Mixture of lipases could be used to obtain higher conversion of canola oil to biodiesel.