Abstract: In this study, WPI hydrogel carriers with pH of 3.0, 5.0, 6.0, 7.0, 8.0 and whey protein isolate (WPI) concentration of 0.2%, 0.3%, 0.4%, 0.5%, 0.6% (w/v) were developed for the delivery of kaempferol (Kae). The state of WPI-Kae hydrogels was observed, and the particle size, potential, encapsulation efficiency and other parameters were analyzed to determine the optimal encapsulation conditions. In addition, the interaction mechanism between WPI and Kae was studied, and the gastrointestinal sustained-release effect of WPI hydrogels on Kae was evaluated. The results of the WPI-Kae hydrogel state, particle size, Zeta potential and encapsulation efficiency indicated that a good uniform and stable WPI-Kae hydrogel was formed when the WPI concentration was 0.5% (w/v) at pH 7.0. Under this condition, the Zeta potential, particle size and encapsulation efficiency of WPI-Kae hydrogels were −37.2 mV, 258.6 nm and 80.61%, respectively. Meanwhile, the hydrogel particles were spherical with uniform distribution. Spectroscopic studies showed that the characteristic peaks of Kae disappeared after self-assembly with WPI, which indicated that Kae was successfully encapsulated by WPI hydrogels, and the formation of hydrogels changed the secondary structure of the protein. The results of in vitro digestion exhibited that WPI hydrogels improved the digestive stability of Kae, and the retention rate reached 58.1% after 2 h of digestion in the small intestine. Molecular docking results showed that Kae formed hydrogen bonds with Asn88, Met107 and Ser116 of WPI, and formed hydrophobic interactions with Leu39. This study will provide a theoretical basis for the construction of Kae delivery systems.