Abstract: To produce micro-biodiesel from microbial oil, transesterification conditions were studied using oil from Rhodotorulaglutinis as raw material using unsaturation index of fatty acid (UIFA)as index. The result showed that the conditions of:reaction temperature 55℃, molar ratio of methanol to oil 40:1, H2SO4 75% based on oil weight, and time more than 6h. These conditions are favourable to accomplish the process of tansesterification sufficiently.