Biodiesel is a clean and renewable fuel, which is considered as the best alternative to diesel fuel, but the feedstock contributes more than 70% of the cost. The most important constituent essential for biodiesel development is to explore cheap feedstock with high oil content. In this work, we found novel non-edible plant seeds of Koelreuteria paniculata (KP) with high oil contents of 28–30 wt.% and low free fatty acid contents (0.91%), which can serve as a promising feedstock for biodiesel production. KP seed oil can convert into biodiesel/fatty acid methyl esters (FAMEs) by base-catalyzed transesterification with the highest biodiesel production of 95.2% after an optimization process. We obtained the optimal transesterification conditions, i.e., oil/methanol ratio (6:1), catalyst concentration (0.32), reaction temperature (65 °C), stirring rate (700 rpm), and reaction time (80 min). The physico-chemical properties and composition of the FAME were investigated and compared with mineral diesel. The synthesized esters were confirmed and characterized by the application of NMR (1H and 13C), FTIR, and GC-MS. The biofuel produced from KP seed oil satisfies the conditions verbalized by ASTM D6751 and EN14214 standards. Accordingly, KP source oil can be presented as a novel raw material for biofuel fabrication.