Energy security, socioeconomic aspect, and environmental concern are the major arising factor to be considered while selecting the source of energy in today’s word including Ethiopia. The rising cost of petroleum fuels, depletion of reserves and strict world protocols on exhaust emissions have also necessitated the need for replacement of petroleum fuels with less polluting, readily available and renewable sources of fuels for automobile and industrial engines. This enforces the worlds to look over the biofuel as a major substitute for mineral fuel sources. Production of biodiesel from non-edible feedstocks is attracting more attention than in the past, for the purpose of manufacturing alternative fuels without interfering with the food chain. Among those non-edible feedstocks, croton macrostachyus seed is another option and which is newly identified feedstock which is indigenous in Ethiopia. The seed was collected from the southeastern part of the country and solvent oil extraction method was selected to extract oil from croton seed. The study aimed at optimizing the biodiesel production process experimentally by varying catalyst concentration, reaction temperature and the molar ratio of methanol to oil. These process parameters were optimized by using design expert software. The optimum conversion efficiency of croton oil to fatty acid methyl ether (FAME) obtained was 96% at the optimal condition of 6:1 methanol to oil ratio, 1% catalyst loading, and 50oC reaction temperature. The properties of croton methyl-ether which were determined fell within the recommended standards. Oil content of the seed was found 53.34% which is beyond estimated value before this study. The main physicochemical properties of biodiesel production from croton oil were characterized and evaluated according to the minimum requirement of international standard (ASTM and EN). It was found that croton biodiesel viscosity 4.613mm2/s, density 0.854g/cm3, calorific value 39.889MJ/kg, flash point 220oc, iodine value 107.084mgI/gm and FFA 0.435 mg KOH/gm. The results indicated that the properties of fatty acid methyl ether obtained from Croton macrostachyus seed were agreed with the standard specification of the ASTM and EN. In addition, the oil content of croton seed was observed that 53.34% which is beyond estimated value before. Finally from this study croton seed was discovered as new biodiesel feedstock and extend the choice of feedstock.