Phytosterol (PS) is a steroid, and its bioavailability can be enhanced by interacting with protein in the C-24 hydroxyl group. The interaction between sterols and amino acid residues in proteins can be enhanced by enzymatic hydrolysis. Phytosterol and whey insulation hydrolysates (WPH1-4) fabricated by the Alcalase enzyme at different enzymatic hydrolysis times were selected as delivery systems to simulate sterol C-24 hydroxyl group interaction with protein. Increasing hydrolysis time can promote the production of β-Lg, which raises the ratio of β-turn in the secondary structure and promotes the formation of interaction between WPH and PS. The correlation coefficient between hydrogen bonds and encapsulation efficiency (EE) and bioaccessibility is 0.91 and 0.88 (P < 0.05), respectively, indicating that hydrogen bonds of two components significantly influenced the combination by concealing the hydrophobic amino acids and some residues, which improved PS EE and bioavailability by 3.03 and 2.84 times after PS was combined with the WPI hydrolysate. These findings are expected to enhance the absorption of PS and other macromolecules by protein enzymatic hydrolysis to broaden their applications for food.