Three heterotrophic microalgae identified as Scenedesmus sp. Y5, Scenedesmus sp. Y7 and Chorellasp. Y9 were isolated and screened from natural water based on biomass yield and lipid productivity. Fatty acids’ composition analysis showed that both Y5 and Y7 mainly contained C16:0, C18:1 (n - 9), C18:2 (n - 6) and C18:3 (n - 3) and Y9 mainly contained C16:0, C18:0 and C18:2 (n - 6), suggesting that these microalgae can be ideal feedstock for biodiesel. Considering the specific growth rate and lipid productivity, the culture conditions were optimized for Scenedesmus sp. Y5, Scenedesmus sp. Y7 and Chorellasp. Y9. Based on the optimization of cultural conditions, all of these three microalgae were tested in fed-batch fermentation, and their biomass productivities were 4.960 g·L-1·d-1, 5.907 g·L-1·d-1 and 4.038 g·L-1·d-1; lipid productivities reached 1.5120 g·L-1·d-1, 1.233 g·L-1·d-1 and 0.8112 g·L-1·d-1, respectively.