In this paper, based on the coupling calculation of Simulink software and GT-Power software, an Optimizer model method was proposed for a 16V265H diesel engine to study the effects of different ratios of biodiesel (B0, B10, and B20) on the performance of a 16V265H diesel engine at 1000 rpm and 75% load. Firstly, the four parameters of diesel engine power, BSFC, soot emission, and NOx emission were taken as the result variables of the optimization model. Taking the intake and exhaust timing of the diesel engine as the independent variable of the optimization model, the maximum power, minimum BSFC, and minimum diesel engine emission were studied and analyzed. Finally, the performance parameters were comprehensively analyzed to determine the best intake and exhaust valve timing. Moreover, based on the model optimization, the diesel engine’s BSFC and power performance were compared, and the optimal intake timing scheme for the diesel engine with different biodiesel ratios at 75% operating conditions was obtained. The results showed that the maximum power, optimum BSFC, and minimum emissions of the 16V265H diesel engine with different ratios of biodiesel and different intake valve timing angles were also different. Under 75% load conditions, the BSFC reduction rate was up to 6.32%, and the power increase rate was up to 5.87%. In addition, by optimizing the model with B10 biodiesel and the intake valve timing close to 202°CA and the exhaust valve timing close to 98°CA, the diesel engine had the lowest NOx emission; with B10 biodiesel and the intake timing at 180°CA, the diesel engine had the lowest BSFC; and with B10 biodiesel and the intake valve timing close to 179.5°CA, the diesel engine had the maximum power. In conclusion, the diesel engine is best with B10 biodiesel. This research method can provide a reference for implementing variable intake system technology for the 16V265H diesel engine.