Enzymatic production of biodiesel from canola oil using self-made covalently immobilized lipase from Candida antarctica (CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) on epoxy-functionalized silica as low cost catalyst was investigated. In this optimization study, the effect of water, t-butanol and water adsorbent content on the yield of fatty acid methyl ester (FAME) was considered. Complete conversion to FAMEs was achieved under optimum conditions for CALB immobilized on epoxy-functionalized silica (Silica-CALB); 30% (w/w) t-butanol by substrate weight, reaction time of 96 h, 50 °C and molar ratio of methanol to oil 3:1, which was added to the reaction mixture in three steps. In general by adding t-butanol to the reaction medium, the conversion of oil to FAME increased for RML immobilized on epoxy-functionalized silica (Silica-RML) and 50 wt.% t-butanol by substrate weight gave the best yield. TLL immobilized on epoxy-functionalized silica (Silica-TLL) reach to 100% yield at 10% t-butanol. Water suppresses the methanolysis reaction catalyzed by Silica-CALB but significantly increased FAME yield for Silica-TLL and Silica-RML. It was observed that at high water adsorbent amounts (more than 55%, w/w), conversion to FAME decreased possibly due to mass transfer limitation. The immobilized TLL was quite stable and can be reused for 16 cycles without significant loss in activity (95%). The immobilized preparations of RML and CALB also presented a good reusability, keeping 85% of their initial activities after 16 cycles of the reaction.