Catalyst development is one of the most challenging aspects in biodiesel production due to the compatibility issue with the feedstocks and catalyst poisoning upon exposure to process conditions. In the present research, chemoenzymatic catalyst was prepared by impregnation of magnesium aluminate (MA) on alumina support, modifying the support using activating agent, and immobilization of Candida rugosa lipase (CRL) onto the support. The optimum immobilization condition was resulting in 85.07 % of activity recovery of immobilized lipase, equivalent to 30.53 % of immobilization efficiency. The catalysts were characterized through XRD, FTIR, FESEM-EDX, NAA and CO2-TPD confirming the mixture of AlO(OH), magnesium aluminate (MA), (MgNO3)2 and Mg2+ on the support. CRL/L-lysine/MA showed remarkable improvement in thermal stability (40 − 70℃), solvents tolerance (ethanol, methanol, isopropanol, tert-butanol), and storage stability at 30 ℃, as compared to free CRL. CRL/L-lysine/MA successfully produced biodiesel yield up to 87.10 % at 200.71 rpm of agitation speed, 13.58 % (w/w) of water content, 10 h reaction time, 15 % (w/w) of catalyst loading, at 50 ℃, using 1:12 of oil to methanol molar ratio, and retaining 46.60 % of biodiesel yield after six cycle. The CRL/L-lysine/MA demonstrated novel catalysts characteristics comprising chemical and biological active species on a single support for biodiesel production from waste cooking oil (WCO).