Waste frying oil, which not only harms people's health but also causes environmental pollution, can be a good alternative to partially substitute petroleum diesel through transesterification reaction. This oil contained 8.8 % of free fatty acids, which cause a problem in a base-catalyzed process. In this study, synthesis of biodiesel was efficiently catalyzed by the covalently immobilized Talaromyces thermophilus lipase and allowed bioconversion yield up to 92 % after 24 h of reaction time. The optimal molar ratio was four to six parts of methanol to one part of oil with a biocatalyst loaded of 25 wt.% of oil. Further, experiments revealed that T. thermophilus lipase, immobilized by a multipoint covalent liaison onto activated chitosan via a short spacer (glutaraldehyde), was sufficiently tolerant to methanol. In fact, using the stepwise addition of methanol, no significant difference was observed from the one-step whole addition at the start of reaction. The batch biodiesel synthesis was performed in a fixed bed reactor with a lipase loaded of 10 g. The bioconversion yield of 98 % was attained after a 5-h reaction time. The bioreactor was operated successfully for almost 150 h without any changes in the initial conversion yield. Most of the chemical and physical properties of the produced biodiesel meet the European and USA standard specifications of biodiesel fuels.