Utilization of low-cost raw materials for the bio-based chemical production, such as carotenoids, by the co-culture of Rhodotorula glutinis and Chlorella vulgaris has recently become an attractive option. In this study, the primary nutrients of starch wastewater were analyzed, which were used for carotenoid production by the co-culture strategy in a 5-L fermenter around 4000 Lux light intensity. Synergistic effect of gas utilization revealed that the two species could build up the beneficial balance on mutualism. The maximum carotenoid productivity and COD removal efficiency were 12.34 mg/L and 79.6%, respectively, which were higher than those of monoculture yeast (8.31 mg/L and 54.1%). The organic acids, amino acids, and sugar removal efficiencies were increased by 85%, 31%, and 44%, respectively, and more than three kinds of carotenoids were identified compared with those of monoculture yeast. The results demonstrated that the co-culture strategy of two different nutritional microorganisms could significantly improve carotenoid productivity and COD removal efficiency.