This study focuses on the optimization of biodiesel synthesis using non-edible hempseed oil as the feedstock. The Response Surface method was used to find the best methanol:oil molar ratio, catalyst concentration, reaction temperature, and reaction duration for the transesterification process. The center composite design experimental design was used to make the design. A total of 30 cycles were conducted to adjust the four parameters at five different levels in order to optimize the biodiesel production process. It was found that the best conditions for transesterification of hempseed oil were a KOH catalyst concentration of 0.80 wt%, a molar ratio of 7.41:1, a reaction time of 62.83 minutes, and a reaction temperature of 61.92 °C. Under these optimized reaction conditions, the predicted biodiesel yield was 95.57%, while the experimental yield was 95.24%. The biodiesel produced using the optimized parameters was analyzed for its properties, and the findings demonstrated that it met the requirements of EN14214, a standard for biodiesel quality. The optimization of the biodiesel synthesis process using non-edible hempseed oil contributes to the exploration of alternative and sustainable feedstocks for biodiesel production. The values of the produced biodiesel within the standard range demonstrate its suitability for commercial applications and strengthen the potential of hemp seed oil as a suitable raw material for biodiesel production.