Abstract: To explore the optimum nitrogen source for outdoor cultivation of Desmodesmus sp. T28-1 and Desmodesmus sp. NMX451, this study investigated effects of different nitrogen sources (urea, nitrate, ammonium and combined urea and nitrate) on the growth and lipid production and evaluated the best mixed nitrogen source for outdoor cultivation. The results of indoor cultivation showed that both T28-1and NMX451 had the lowest lipid content under urea and the lowest biomass productivity under ammonium. NMX451 obtained significantly higher lipid productivity under the combination of urea and nitrate than any other nitrogen source. Both T28-1 and NMX451 obtained high biodiesel quality under mixed nitrogen source, which met the standards of America, Europe and China. Results of cultivating the two microalgae with mixed nitrogen source in an outdoor 140 L photobioreactor showed that NMX451 had higher lipid content and lipid productivity and even better biodiesel quality than cultivation indoor. These results suggest that mixed nitrogen source can be used to produce microalgae-based biodiesel production outdoors, and NMX451 had better biofuel production under this condition than T28-1.