In this study, biodiesel was produced from Parkia biglobosa oil via optimization of transesterification reaction conditions, (methanol to oil ratio, catalyst concentration, reaction temperature, and reaction time) under sulphuric acid catalyst (H2SO4). The oil was first extracted from Parkia biglobosa seeds using the Soxhlet extraction method. The physicochemical properties of the biodiesel were analysed and then compared to international standards. Subsequently, the oil was then used to produce biodiesel at optimized transesterification reaction conditions. The free fatty acid (FFA) content of the oil was 1.61% w/w, while the saponification value (mgKOH/g) was 191.65. The maximum yield (percentage weight) of the biodiesel produced was 93.4% at the maximum transesterification conditions of methanol-to-oil molar ratio of 6 : 1, sulphuric acid catalyst amount of 3 wt%, reaction temperature of 65°C, and reaction time of 1.5 h. The biodiesel produced was within the limits of international standards as per the specification by ASTM D6751 (American standard), EN 14241 (European standard), and Ghana Standard Authority. It was therefore recommended that biodiesel from Parkia biglobosa seed oil under acidic catalytic condition is a potential new substitute for petroleum diesel for commercialization purposes.