Chalcone isomerase (CHI) is an important enzyme involved in the biosynthesis of flavonoids, one that is crucial in both plant defense and human health. Although many CHI genes have been previously identified, the function of CHI-like genes in soybean remains unclear. In this study, we cloned the CHI-like genes GmCHI4A and GmCHI4B (GmCHI4s) in soybean. The real-time quantitative polymerase chain reaction showed that GmCHI4s were expressed primarily in soybean root, but were also present in other tissues, including the stem, leaf, and seed with a low expression level. Overexpression of GmCHI4s was able to significantly improve some beneficial traits of the transformed hair roots of cotyledon or composite plants under salt stress conditions. Root length, root wet weight, and the underground biomass was increased, and the elevation of MDA content was inhibited under 100 mmol L⁻¹ or 150 mmol L⁻¹ NaCl treatment. Leaf chlorophyll content was elevated in overexpressed GmCHI4A composite plants under 150 mmol L⁻¹ NaCl treatment. The expression levels of salt-stress-related genes GmSOD1, GmAPX1, GmSOS1, and GmNHX1 were significantly upregulated in overexpressed GmCHI4 hairy roots compared to that in empty-vector-expressed hairy roots. The above results indicated GmCHI4s’ potential action against salt stress. Furthermore, overexpression of GmCHI4A and GmCHI4B increased the total isoflavone content by six times and three times, respectively. Glycitin and glycitein levels were significantly elevated in the overexpressed GmCHI4A hairy roots, while glycitin, genistin, daidzein, and genistein were significantly increased in overexpressed GmCHI4B hairy roots. This study identified a new function of the CHI-like gene, as well as providing a new selected gene for salt tolerance and isoflavone improvement using biotechnological approaches in soybean.