The present research work explores biodiesel production from one of the novel non-edible feedstocks viz. Cyprinus carpio fish oil. Production of biodiesel from this oil was successfully performed through base-catalyzed transesterification assisted by a co-solvent. Hexane and potassium hydroxide (KOH) were chosen as the co-solvent and the catalyst, respectively. The experimental parameters included in the optimization process were type of co-solvent, type and concentration of the base catalyst, methanol to oil molar ratio, hexane to methanol volume ratio, temperature and time. The maximum yield of biodiesel from C. carpio fish oil (98.55 ± 1.02% ∼ 97.24% w/w ester content) was obtained at 0.60% KOH w/w, 5:1 methanol to oil molar ratio, 1.5:1 hexane to methanol volume ratio, 50 °C reaction temperature and 30 min of reaction. The Fourier Transform Infrared spectroscopy and thin layer chromatography were used to ensure the conversion of fish oil into biodiesel. The biodiesel properties were within the recommended biodiesel standards as prescribed by ASTM D 6751 and EN 14214. The most remarkable features of C. carpio fish oil biodiesel are the flash point and viscosity which are close to those of petro diesel. Moreover, blends of biodiesel and petro diesel were complied with the limits prescribed in the ASTM D7467 standards. Hence, the oil is a potential non-edible feedstock for biodiesel production. Co-solvent transesterification of C. carpio fish oil was found to follow first order kinetics and the activation energy was calculated to be 32.46 kJ/mol. However, it was concluded that the use of co-solvent within the reaction medium enhanced yield and properties of biodiesel comparing with that produced via nonsolvent process.