Soybean isoflavones, natural phytoestrogens within the flavonoid family, exhibit diverse physiological benefits such as anticancer, antioxidant, and cardioprotective properties. Yet, the underlying biosynthetic pathways remain unclear. Research is required to get better knowledge of soybean isoflavone production and its potential uses. Our work thoroughly examined the R2R3-MYB subclass in soybean and discovered a new MYB transcription factor, GmMYB3a, which shares significant similarities with Arabidopsis MYB genes and regulates isoflavone biosynthesis. Our study reveals that GmMYB3a localizes to the nucleus and membrane, concurs with its potential involvement in the biosynthesis of isoflavones. Our analysis also indicated a synergistic expression pattern between GmMYB3a and seed development, thereby creating the hypothesis that it has a critical role in the regulation of isoflavone synthesis. Transgenic experiments further demonstrated that GmMYB3a positively regulates isoflavone biosynthesis and leads to its overexpression. GmMYB3a has been implicated in abiotic stress responses, affecting soybean stress tolerance. RNA sequencing analysis revealed that GmMYB3a regulates downstream genes involved in isoflavone, flavonoid, and phenylalanine metabolism, especially the key chalcone synthase genes, CHS7 and CHS8. Moreover, GmMYB3a was shown to be tightly associated with GmCHS7 and GmCHS8 expressions, potentially regulating them directly. Yeast two-hybrid screening identified GmMYB3a interacting proteins crucial for the synthesis of physiologically active substances and abiotic stress responses. Our results increase knowledge of the regulatory mechanisms of GmMYB3a and establish a molecular network involving GmMYB3a, GmCHS7, and GmCHS8, thereby offering novel strategies for improving soybean quality and stress-tolerant breeding.