Biodiesel has been accepted as a clean and an eco-friendly green diesel fuel by the entire world. In India, various non-edible oils have been tested for exploring their suitability as a fuel in diesel engines. In the north eastern states of India, many oil bearing seeds such as Koroch (a variety of Pongamia glabra), Nahar (Mesua ferrea), Terminalia (Terminalia belerica Robx), Kutkura (Meyna spinosa Roxb), Amari (Amoora Wallichii King), Yellow oleander (Thevetia peruviana) and others are found in abundance. In this article, the Yellow oleander seed oil (YOSO) has been investigated for biodiesel production and characterization. The oil content in Yellow oleander seed is found to be 63.87%. The free fatty acid (FFA) content in YOSO is measured, and is found to be 32.0%; hence the two-step acid-base catalysis transesterification process has been adopted for producing biodiesel from the YOSO. YOSO contains 5.03% palmitic, 6.92% stearic, 48.14% oleic and 31.37% linoleic acid. The density, calorific value and kinematic viscosity of Yellow oleander fatty acid methyl ester (YO-FAME) are 879.7 kg/m3, 40.159 MJ/kg and 4.63 mm2/s respectively. Most of the fuel properties of YO-FAME meet ASTM D6751 and EN 14214 biodiesel standards. The YO-FAME exhibits a low sulphur content of 13.0 ppm and a high cetane number of 58.3. Fire point and pour point of YO-FAME were found to be 158°C and 5°C respectively. The physio-chemical properties of YO-FAME has been compared with FAME of Yellow oleander, Ratanjot (Jatropha curcus), Terminalia (Terminalia belerica Robx.) and Nahar (Mesua ferrea).