Objective To investigate the formulation process of Gleditsia sinensis polysaccharide microemulsion and the hydrophobic interactions between the microemulsion and quercetin.Methods A pseudo-ternary phase diagram was drawn through compatibility experiments to optimize the formulation of the G. sinensis polysaccharide microemulsion. Based on the pyrene fluorescence probe technique, the effect of quercetin concentration on the I1/I3 value in the microemulsion was examined to explore the hydrophobic and polar interactions between quercetin and the carrier in the G. sinensis polysaccharide microemulsion system.Results The prepared G. sinensis polysaccharide microemulsion was of the oil-in-water (O/W type). The optimal formulation consisted of 35.4% Tween 80-Span 80 (mass ratio 3∶2), 17.7% anhydrous ethanol, 13.3% liquid paraffin, and 33.6% G. sinensis polysaccharide solution (5 mg/mL). After ultrasonic treatment for 10 min, the resulting microemulsion exhibited a smaller particle size and a larger absolute ζ-potential, indicating better stability. Within a certain range of quercetin concentrations, longer ultrasonic treatment led to a lower relative molecular mass of the G. sinensis polysaccharide and decreased sensitivity of the I1/I3 value of the microemulsion.Conclusion The preparation process of the O/W microemulsion using G. sinensis polysaccharide is simple. The microemulsion formed from low-relative-molecular-mass G. sinensis polysaccharide obtained via ultrasonic treatment significantly suppresses the sensitivity of hydrophobic interactions to quercetin concentration, which is beneficial for achieving stable and controlled release of quercetin in the gastrointestinal environment.