Isoflavones are a sub-class of phenylpropanoids having health benefits and a role in plant defence and plant-rhizobium interaction. Isoflavone conjugate hydrolysis is crucial in determining the bioactivity and bioavailability of these isoflavones inside the human body. This study examined the different characteristics of soy isoflavone conjugate hydrolysing β-glucosidase (GmICHG) to explore its potential for isoflavone bioavailability enhancement. We cloned the full-length GmICHG cDNA from the soybean seedling roots from the DS2706 variety of 1545 bp. The bioinformatics analysis revealed secretion and glycosylation of this protein. The evolutionary relatedness of this gene to the other glucosidases interestingly had related sequences outside the Papilionaceae family. The protein had a pI above neutral of 7.62 and optimum pH of 6.0, indicating its activity in the extracellular acidic environment. The GmICHG gene expression at three stages of seedling roots gradually rose to 1.84 ± 0.54 fold and a concomitant increase in the β-glucosidase activity. The enzyme kinetics of GmICHG showed a K_m of 6.38 mM and V_max of 2.82 U/ml and an optimum temperature of 40 °C. These hint that soy ICHG can be a potent candidate for the isoflavone bioavailability enhancement by hydrolysing their β-glycosidic bonds.