Abstract: To construct a heterotrophic axenic system of Chlorella sorokiniana FZU60, the symbiotic bacteria were isolated and identified, and the effects of eight common antibiotics (cefotaxime, kanamycin, neomycin, ampicillin, streptomycin, tetracycline, chloramphenicol, and erythromycin) on the cell growth of symbiotic bacteria and C. sorokiniana FZU60 were investigated. Results showed that the main symbiotic bacteria were Bacillus (78.76%) and Methylobacterium (3.14%). Besides, 10 mg/L cefotaxime, 10 mg/L tetracycline, 100 mg/L kanamycin, and 100 mg/L erythromycin showed excellent inhibitory effect on these two symbiotic bacteria but no obvious effect on the cell growth of C. sorokiniana FZU60. Based on the above-mentioned results, the inhibition effect of different antibiotic combinations on symbiotic bacteria of C. sorokiniana FZU60 was further investigated. It was found that the heterotrophic axenic system could be successfully constructed by using the combination of 10 mg/L cefotaxime, 10 mg/L tetracycline, and 100 mg/L kanamycin. Moreover, C. sorokiniana FZU60 could maintain stable and axenic growth after three generations. The experimental results would provide theoretical reference and experimental basis for the preparation of axenic microalgal seed for high-cell-density heterotrophic cultivation of C. sorokiniana.