Phenolic compounds in mulberry leaves can reduce the digestion and absorption of fat to achieve weight loss; however, the mechanism of their interaction with lipase remains unclear. The inhibitory effects of 12 mulberry leaf phenolic compounds on lipase were investigated through in vitro lipase activity assays. Hyperoside and rutin, which share the same core structure, were found to have stronger inhibitory effects on lipase activity. The hyperoside and rutin inhibition mechanism on lipase was investigated using fluorescence spectrum analysis, FT-IR analysis, and molecular docking simulation. Fluorescence spectrum analysis showed that the quenching mechanism of lipase by hyperoside and rutin was static quenching. The binding constant of lipase with hyperoside at 25 ℃ was 365.79×1011 L/(moL•s), and the corresponding value at 30℃ was 136.27×1011 L/(mol•s). These values were found to be higher than those observed for rutin (4.50×1011 and 2.08×1011 L/(mol•s), respectively). FT-IR analysis revealed that the binding of hyperoside and rutin with lipase changed the secondary structure of lipase. Moreover, molecular docking analysis demonstrated that hyperoside and rutin were bound to lipase through hydrogen bonding and hydrophobic interaction, thus inhibiting lipase activity. In addition, the ΔG value of lipase binding with hyperoside (−29.21 kcal/mol) was lower than that of lipase binding with rutin (−20.89 kcal/mol). This study elucidated the inhibitory mechanism of phenolic compounds in mulberry leaves on lipase. These findings establish a theoretical foundation for further research and development of mulberry leaves as functional food for weight management.