The use of biofuel has been researched and announced by scientists to bring benefits in terms of environmental protection. However, studies continue to be conducted to achieve stable results and confirm biofuels as an effective alternative fuel for internal combustion engines. In this study, the fuel used is biodiesel derived from fish oil and conventional diesel fuel. Specifically, the test fuel is conventional diesel mixed with fish oil biodiesel in different ratios, including B0, B10, B20, B30, B40, and B50 (respectively 0%, 10%, 20%, 30% 40%, and 50% biodiesel in the mixture). Research was carried out by both simulation (from B0 to B50) and experiment (from B0 to B30) on a single cylinder common rail diesel engine. Test modes were at 25%, 50%, and 75% load respectively at maximum torque (at 1400 revolutions per minute (rpm)) and minimum fuel consumption (at 2200 rpm). Compared with B0, the average reduction in brake power of the biodiesel decreased relative to the proportion of biodiesel in the fuel mixture. Specific fuel consumption of the brakes (BSFC) and NOx emissions increased with decreasing emissions of smoke, hydrocarbon (HC), and carbon monoxide (CO) emissions when the biodiesel ratio increased.