Abstract: 【Objective】This study optimized the culture medium for Chlorella vulgaris and Scenedesmus obliquus， which provided reference for improving the growth rate of mixed greenalgae and promoting the application of mixed culture technology of freshwater beneficial algae.【Method】The concentration ranges of nutrient salt that suitable for the proliferation of C. vulgaris and S. obliquus in BG11 medium were screened by single factor test. And the nutrient factors that significantly affected the growth of the mixed green algae were determined by Plackett-Burman test. By using response surface methodology，the optimal concentrations of the main nutrient salts in BG11 medium were optimized. Then，confirmatory experiment of the mixed green algae culture was performed.【Result】Relatively suitable concentrations of 7 nutrient salts for the mixed green algae growth were determined by single factor test. Using the Plackett-Burman test， 3 nutrient factors，Na₂CO₃， K₂HPO₄ and NaNO₃， which significantly impacted the growth of mixed green algae most were determined via Plackett-Burman test. The results of response surface methodology analysis showed that the strong interaction between NaNO₃ and K₂HPO₄ had a significant effect on the cell density of mixed green algae（P＜0.05，the same below）. In the optimized medium， NaNO₃， K₂HPO₄ and Na₂CO₃ were 1600.00， 50.85 and 32.39 mg/L，respectively. Maximum light conversion efficiency in the photosynthesis PSII system of the mixed green algae in the optimized medium was significantly improved. The cell density of mixed green algae in the optimized medium group peaked at 36.75×10⁶ cells/mL， which was significantly increased by 24.28% than the control group. Furthermore，the cell density rate of C. vulgaris and S. obliquus was basically 1.00∶1.00 throughout the culture cycle.【Conclusion】The mixed green algae culture medium of C. vulgaris and S. obliquus optimized by response surface methodology can realize the high density culture of mixed green algae，which can be used as a reference for the large scale application of mixed culture technology of freshwater beneficial algae；the optimal concentrations of Na₂CO₃，K₂HPO₄ and NaNO₃ in the culture medium of two mixed green algae are 32.39， 50.85 and 1600.00 mg/L respectively.