Fresh flavor is the key factor of the taste quality of vegetable soybean, and its formation is closely related to organic acids. It is of great practical significance to study the synthesis mechanism of organic acids for the quality improvement of vegetable soybean. In this study, candidate genes GmALMT8, GmIF7GT5 and GmAP, which are significantly related to malic acid content, were used in vegetable soybean hairy root system to explore their functions in regulating malic acid content. The results showed that GmALMT8 gene expression and malic acid content in GmALMT8-OE hairy root were significantly higher than that in no-load control hairy root.. In view of the reported malic acid transport function of ALMT family genes, it is speculated that GmALMT8 gene in vegetable soybean may have similar functions and play an important role in regulating malic acid content. The genetic transformation of Arabidopsis thaliana further proved that stable expression of GmALMT8 could increase malic acid content, and it was clear that GmALMT8 gene had a biological function of regulating malic acid content in vegetable soybeans, which laid a theoretical foundation for the study of molecular regulatory mechanisms of organic acid accumulation.