This study explores the feasibility of producing biodiesel from dehulled orange seed oil, a non-edible agro-industrial byproduct with significant potential as a renewable energy feedstock. The research aims to enhance biodiesel yield through the optimization of transesterification process parameters using Response Surface Methodology (RSM). Dehulled orange seeds were processed to extract oil, after which transesterification was carried out using methanol. Five key process factors—reaction temperature, reaction time, catalyst concentration, methanol-to-oil molar ratio, and agitation speed—were systematically varied based on a central composite design to assess their individual and interactive effects on biodiesel yield. Statistical analysis indicated that all variables influenced conversion efficiency, with methanol ratio and catalyst concentration exerting particularly strong effects. The quadratic model developed showed high predictive accuracy and statistical significance, confirming its suitability for optimization. The optimal reaction conditions were identified as a temperature of 75 °C, reaction time of 150 minutes, catalyst concentration of 5 wt%, methanol-to-oil molar ratio of 12:1, and agitation speed of 350 rpm. Under these conditions, the biodiesel yield reached 95.23%, demonstrating efficient conversion and validating the optimization strategy. The physico-chemical characteristics of the produced biodiesel further complied with standard fuel specifications, underscoring its suitability as a renewable fuel. Overall, the results affirm that dehulled orange seed oil is a viable and sustainable feedstock for biodiesel production. The optimized process not only achieves high yields but also adds value to agricultural waste streams, contributing to cleaner energy alternatives and supporting circular bioeconomy initiatives. This study highlights the importance of exploring non-edible oils for biodiesel production to reduce competition with food resources and promote environmental sustainability.