Biodiesel production has been studied using non-edible seed oil from Thevetia peruviana. The non-edible oil from Thevetia peruviana seed could be used as a feedstock for biodiesel manufacturing. In this paper, the production of biodiesel utilizing Thevetia peruviana Seed Oil (TPSO) has been investigated. The ripe seeds of Thevetia peruviana were gathered from China and Pakistan, and after drying, its oil was extracted using the Soxhlet procedure. The seeds' highest oil content was discovered to be between 48-62%. Investigations were done into the TPSO's physicochemical characteristics. Transesterification was used to make biodiesel. The methanol to oil molar ratio, the duration, temperature, rate of stirring, and the amount of catalyst loaded were all studied in order to optimize the biodiesel synthesis process and get the highest yield. Under the ideal conditions, which included a methanol-to-oil molar ratio of 6:1, a catalyst loading of 0.42 weight percent, a temperature of 70°C, a stirring rate of 700 rpm, and an 80-minute reaction period, the biodiesel yield was 98.4%. Gas Chromatography (GC) analysis was used to identify the fatty acid configuration of the oil. Nuclear Magnetic Resonance Spectroscopy (NMR, 1H NMR and 13C NMR) and Fourier Transform Infrared Spectroscopy (FTIR) were used to characterize the biodiesel product. The ASTM & EN test technique were used to determine the biodiesel's fuel characteristics. Thevetia peruviana seed oil is used to make biodiesel that satisfies the requirements of ASTM D6751 and EN standards. As a result, this seed oil is a possible feedstock for the production of biodiesel.