The invention discloses a method for improving biomass and oil accumulation of oleaginous microalgae Scenedesmusdimorphus by adopting a two-stage culture strategy of co-culture and nitrogen deficiency, and belongs to the technical field of microalgae bioenergy. The invention adds a certain concentration of organic carbon source glucose to the autotrophic culture medium, and carries out concurrent culture with CO2 as the autotrophic carbon source. At the same time, under nitrogen-enriched conditions, after high-density culture of microalgae to a stable stage, the algae liquid is transferred to a nitrogen-deficient medium to continue the culture. When the glucose concentration is 1.2-5.4g/L, the initial pH is 7, and the culture time is 14-16 days, the biomass of microalgae can reach up to 4.5g/L, which is 2.57 times the biomass under autotrophic conditions; nitrogen deficiency Under these conditions, the total lipid content of microalgae reaches the highest of 29.62%, which is 1.6 times that of the nitrogen-enriched stage. By adding organic carbon sources and providing nitrogen-deficient conditions for two-stage culture, the biomass of microalgae can be effectively increased and oil accumulation can be promoted, which is of great significance to the research of large-scale production of bioenergy by microalgae.