The programmed death-ligand 1 (PD-L1) protein plays a key role in immune responses. Scutellarin (SCU), as a flavonoid, has a variety of bioactivities. In this study, the human PD-L1 was obtained by expression and purification, and the interaction mechanisms between PD-L1 and SCU were revealed through multi-spectroscopy and computer simulation. Fluorescence data indicated that the quenching of PD-L1 by SCU was mainly static quenching, and primarily driven by hydrogen bonding and van der Waals forces. The binding constant (K