Microalgae have characteristics similar to higher plants, and recent research has shown that phytohormones can influence the metabolic and physiological processes of microalgae. Utilizing phytohormones to adjust the production capacity of microalgae could improve their position in the industrial raw material supply chain, accelerate the transition to green and sustainable industry, and reduce carbon emissions. This study investigated the effects of six phytohormones, including 6-benzylaminopurine, ethephon, gibberellin, indole acetic acid, naphthalene acetic acid, and salicylic acid, on the growth and biochemical composition of Chlorella vulgaris 31(Cv-31) after 12 days of culturing. The results showed that there were significant variations in the effects of different concentrations and species of phytohormones on the growth and biochemical composition of Cv-31. Among the six phytohormones, 10 mg L− 1 GA was the most effective in promoting growth and pigment accumulation, while 7.5 mg L− 1 SA resulted in a 3.3-fold increase in lipid production compared to the control. However, the phytohormones did not play a significant role in protein accumulation, while ETH and GA were helpful in the accumulation of soluble sugars. This study provides a comprehensive understanding of the effects of phytohormones on the main components of Cv-31 and can serve as a reference for related research and production.