In this paper, hybrid-coprecipitation and combustion methods were used to synthesis MgAl2O4 spinel. Of interest was also to evaluate the effect of different synthesis methods on the characteristics and performance of MgO/MgAl2O4 catalyst in biodiesel production. Physicochemical characteristics of synthesized catalysts were evaluated by XRD, FESEM, surface particle size distribution (SPSD), EDX-Dot Mapping, BET-BJH and FTIR analysis. Moreover, the final performance of the samples was investigated in biodiesel production. The results of the XRD analysis showed that the highest crystallinity and crystal size belonged to the sample synthesized by the combustion method. Furthermore, the FESEM analysis showed that the sample synthesized by the combustion method had a unique morphology and the other samples had almost the same morphology. The BET-BJH analysis indicated that the least surface area and pore volume were related to the sample prepared by the combustion method. However, by perusing the samples in biodiesel production, it was found that the combustion prepared sample had the largest conversion value (95.7%) and great reusability (6 cycles). According to the total analysis, it can be said that the size and shape of the pores as well as the catalyst crystallinity are the most important specifications for biodiesel production.