A heterotrophic mutant of Chlorella sorokiniana MB-1-M12 was evaluated for its ability to produce lutein using organic carbon and nitrogen sources and without light irradiation. In batch fermentation, the maximal lutein content (3.67 mg lutein/g biomass) and productivity (2.84 mg/L/d) could be obtained when cultivated in BG-11 medium with 7.5 g/L glucose, 0.75 g/L urea, pH 7.5 and a C/N ratio of 10. A novel two-stage cultivation strategy that integrates fed-batch and semi-batch operations was applied to enhance the lutein production performance. When growing MB-1-M12 strain in a 5L fermenter using the optimal operation strategies, the maximum biomass concentration, biomass productivity, lutein content and lutein productivity could reach 25 g/L, 4.88 mg/L/d, 5.88 mg/g and 16.2 mg/L/d, respectively. This high lutein productivity could significantly reduce the cultivation time and the associated costs, indicating the potential of using MB-1-M12 strain for heterotrophic lutein production in commercial scale.