•High cell density of Scenedesmus incrassatulus biomass was achieved in heterotrophic culture.•Vitamins were suggested as regulatory factor in enhancing pigment synthesis.•Two stage hetrotrophy/photoinduction process was employed for S. incrassatulus culture.•Two stage hetrotrophy/photoinduction process improved the biomass and lutein productivity. A biomass production process including two stages, heterotrophy/photoinduction (TSHP), was developed to improve biomass and lutein production by the green microalgae Scenedesmus incrassatulus. To determine the effects of different nitrogen sources (yeast extract and urea) and temperature in the heterotrophic stage, experiments using shake flask cultures with glucose as the carbon source were carried out. The highest biomass productivity and specific pigment concentrations were reached using urea+vitamins (U+V) at 30°C. The first stage of the TSHP process was done in a 6L bioreactor, and the inductions in a 3L airlift photobioreactor. At the end of the heterotrophic stage, S. incrassatulus achieved the maximal biomass concentration, increasing from 7.22gL−1 to 17.98gL−1 with an increase in initial glucose concentration from 10.6gL−1 to 30.3gL−1. However, the higher initial glucose concentration resulted in a lower specific growth rate (μ) and lower cell yield (Yx/s), possibly due to substrate inhibition. After 24h of photoinduction, lutein content in S. incrassatulus biomass was 7 times higher than that obtained at the end of heterotrophic cultivation, and the lutein productivity was 1.6 times higher compared with autotrophic culture of this microalga. Hence, the two-stage heterotrophy/photoinduction culture is an effective strategy for high cell density and lutein production in S. incrassatulus.