The invention discloses a method for producing biological diesel oil by two steps of culture of chlorellas, namely, autotrophy and heterotrophy, which belongs to the renewable biological energy field. Concentrated autotrophic algae is put into a fermentation tank to perform heterotrophic growth from the processes of autotrophic culture, cell concentration and fermentation pollution controlling of the chlorellas, to ensure the chlorellas to be synthesized into neutral fat. Effective feeding strategy is established to ensure the fat to be synthesized into optimal after the optimization of the fermentation conditions and the process control. The biomass can reach 108g.L-1, and the grease content can reach 52 percent of cell dry weight. After the culturing is finished, biological diesel oil can be prepared after extracting algal oil and transesterification reaction. The cell concentration technology adopted by the invention can effectively avoid the problem of light restriction during the high density culture process Not only is the carbon dioxide emission reduced, but also the organic carbon source consumption is reduced, thus the preparation cost of the biological diesel oil material is saved. The whole technical line is environmental friendly, high efficient, and can meet the industrial application requirements of the microalgae biological diesel oil.