The viscera of Nile tilapia, fish processing industry waste, was utilized to extract oil and lipase and used as a low-cost feedstock and catalyst, respectively, for producing biodiesel. The investigation aimed to improve the process of extracting oil from Nile tilapia viscera (NTVO) by a wet rendering method, and to use it for manufacturing biodiesel using Nile tilapia viscera lipase (NTVL)-based catalyst. Heating Nile tilapia viscera waste at 80 °C for 60 min was the best condition for oil separation, with 51.49 % oil recovery and 6.30 % yield. NTVO characteristics suggested the suitability for biodiesel production. Optimizing the synthesis of biodiesel from NTVO utilizing NTVL catalyst was also studied. The catalyst dose of 20 kUnits, reaction temperature of 35 °C, water content of 1 %, reaction period of 18 h, and methanol to oil molar ratio of 4:1 were found to provide the greatest biodiesel yield (96.9 %) (p < 0.05). ATR-FTIR analysis confirmed the conversion of NTVO to biodiesel. The physicochemical properties of biodiesel obtained met the requirement specified by international standards, namely EN 14214 and ASTM D 6751. These findings highlighted the potential of NTVO and NTVL as a substrate and catalyst for biodiesel synthesis, which might alleviate environmental pollution and disposal issues.