Background: Honey has been widely used as a traditional vehicle of herbal medicines. Honey behaves as a natural deep eutectic solvent (NADES) containing β-glucosidase; therefore, it can be used for the extraction and bio-activation of the bioactive compounds of herbs. Objectives: This study aims to apply honey (H-NADES) and a sugar-based NADES (S-NADES) for the extraction, analysis, and bioconversion of daidzin from Pueraria candollei var. mirifica (PM) root. Materials and Methods: Various concentrations of H-NADES and S-NADES (water:sucrose:glucose: fructose, 18:3:18:22 by weight) were used as solvents for extraction. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed and validated for monitoring the extraction efficacy. The catalytic reactivity against daidzin of β-glucosidase purified from honey was investigated. Results: Using NADESs as solvents, icELISA was suitable for the reliable determination of daidzin with high sensitivity (1.95–125 ng/mL), specificity (% cross-reactivity ≤ 2.60), and accuracy (98.3-106% daidzin recovery). Daidzin at a concentration of 75.8 ± 3.67 μg/mL was extracted using 50% (v/v) S-NADES, which was the most effective for the extraction compared to H-NADES, water and ethanol. In addition, daidzin was converted to daidzein by honey β-glucosidase. Conclusion: Both S-NADES and H-NADES were useful for the extraction, analysis, and bioconversion of daidzin, and β-glucosidase from honey might enhance the oestrogenic activity and bioavailability of PM phytochemicals.