Biodiesel synthesis through transesterification of used cooking oil or frying oils using the viscera lipase from Nile tilapia (Oreochromis niloticus) as a low-cost feedstock and catalyst, respectively, was optimized. The influences of operating factors, including methanol/oil molar ratio, enzyme loading, reaction time, type of alcohol, water content and reaction temperature on the yield of biodiesel were investigated. The optimized conditions to achieve maximum biodiesel yield were obtained using an enzyme loading of 30 kUnit, a methanol to oil molar ratio of 4:1, a water content of 3%, a reaction temperature of 45 degrees C and a 28 h reaction time. Under these optimal operating conditions, the highest biodiesel yield observed was 96.5%. The attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) was applied to ensure the conversion of used cooking oil into biodiesel. The biodiesel characteristics met the specifications set as prescribed by EN 14214 and ASTM D 6751. The results from this investigation show the viability of economical biodiesel production using byproducts as both source and catalyst. (C) 2020 Elsevier Ltd. All rights reserved.