To study the effects of antibiotic types and concentrations on biomass and relative oil content of Chlorella, and to optimize the contents of peptone and NaCl in culture medium, Chlorella vulgar HDA04 was used as the test algae strain, different concentrations of antibiotics were added into the culture medium. The biomass and oil content were detected and the medium composition was optimized. The results showed that three antibiotics (kanamycin, tetracycline and penicillin) were added to the initial BG11 medium, penicillin had the best optimization effect on Chlorella vulgar HDA04. When the concentration of penicillin, sodium chloride and peptone was 1.0 mg/L, 8 g/L and 1.5 g/L, respectively, the biomass and relative oil content of C. vulgar HDA04 reached the highest, and the highest biomass was 4.26±0.35 g/L, and the highest relative oil content was 27.04±1.13, which was 111% and 692.3% higher than control group (initial BG11 culture medium), respectively. Optimizing the culture conditions of Chlorella vulgar HDA04 lays a theoretical basis for biodiesel production by Chlorella vulgar, which has great significance to the exploration of microalgae in the alpine saline lake area.