Total flavonoid content is one of the most important quality indicators of Ginkgo biloba leaves and its products, but the complex pretreatment procedures and the excessive use of organic solvents complicate the detection process. In response, an innovative and efficient two-phase deep eutectic solvent (DES) system—composed of an acidic hydrophilic DES and a hydrophobic DES—was developed to quantify flavonoids for quality control of G. biloba products. High-performance liquid chromatography (HPLC) detection conditions were optimized, followed by systematic screening and design of the two-phase DES system through adjustments to polarity, hydrophilicity, and acidity. Optimal pretreatment conditions were established through operating condition optimization. This approach enabled simultaneous extraction, deglycosylation, and enrichment of all flavonoids from G. biloba samples into the hydrophobic phase in a single step. Method validation showed strong linearity with a correlation coefficient (R2 > 0.9995). The detection (LOD) and quantification (LOQ) limits were between 0.35 and 0.37 mg/kg, and 0.92 and 0.96 mg/kg, respectively. The method also exhibited high sensitivity and accuracy, with a recovery rate of 96.91–100.39%. A greenness assessment with the ComplexGAPI tool confirmed this method’s eco-friendly advantages over conventional techniques, aligning well with green chemistry and economic principles. Ultimately, this technique effectively measured flavonoid levels in different G. biloba products. The method developed in this work not only enhances the efficiency of simultaneous extraction and deglycosylation by modifying the polarity, hydrophilicity, and acidity of the two-phase DES system, but also offers valuable insights for the advancement of environmentally friendly analysis technology for flavonoids.