In the present study, waste Asimina triloba seeds are proposed as a potential feedstock for biodiesel production, with the optimization of transesterification process parameters aimed at achieving higher yield rates. While previous studies have explored the use of Asimina triloba biodiesel for engine applications, this study stands out as the first to optimize transesterification process parameters for increased yield and to conduct comprehensive fuel characterization alongside storage stability analysis. The transesterification process parameters such as the methanol-oil molar ratio, catalyst volume concentration, reaction process temperature, and reaction process time are optimized by employing the response surface methodology. The response surface methodology analysis revealed that the proposed model is highly significant, with a coefficient of determination approaching unity. To achieve maximum biodiesel yield, the recommended parameters are a methanol-oil molar ratio of 14.7:1, a catalyst concentration of 2.4%, a reaction temperature of 81.3°C, and a reaction time of 138.8 minutes. Under these optimal conditions, the projected yield is 94.35%, aligning closely with experimental observations of 94.4%. The Fourier-transform infrared spectroscopy analysis underscored the substantial presence of carbon-based constituents in biodiesel, while the gas chromatography-mass spectrometry analysis revealed octadic-9,12-dienoic acid as the primary contributor. Furthermore, the stability profile demonstrates an extended stability period, complemented by physicochemical properties that align with the biodiesel standards specified by ASTM.