The purpose of this study was to elucidate the binding of paeonol to human serum albumin (HSA) through spectroscopic methods. The fluorescence quenching of HSA by paeonol was a result of the formation of the HSA–paeonol complex with low binding affinity (K = 4.45 × 103 M−1 at 298 K). Thermodynamic parameters (ΔG○ = –2.08 × 104 J·mol−1, ΔS○ = 77.9 J·mol−1·K−1, ΔH○ = 2.41 × 103 J·mol−1, kq = 9.67 × 1012 M−1·s−1) revealed that paeonol mainly binds HSA through hydrophobic force following a static quenching mode. The binding distance was estimated to be 1.91 nm by fluorescence resonant energy transfer. The conformation of HSA was changed and aggregates were formed in the presence of paeonol, revealed by synchronous fluorescence, circular dichroism, Fourier transform infrared spectroscopy, three‐dimensional fluorescence spectroscopy, and resonance light scattering results.