Two apigenin derivatives Ⅰ[8-(N,N-diethyl)-methylene-amino-4',5,7,-trihydroxy flavone] and Ⅱ[8-(N,N-diethyl)-methylene-amino-4',5,7,-trihydroxy flavone-3'-sulfonic acid] were synthesized via Mannich and sulfonation reaction, and characterized by ESI-MS, 1H-NMR and 13C-NMR Then fluorescence quenching spectra, synchronous fluorescence spectroscopy, ultraviolet spectroscopy combined with correction of inner filter effect, along with molecular docking studies were used to investigate their interaction mechanism with human serum albumin (HSA) at 298 K, 303 K, 308 K. Spectroscopy results indicated that the intrinsic fluorescence of HSA was quenched by forming a complex with the drug. It was due to the static quenching and non-radiation energy transfer, and the binding site was near the subdomain IIA of HSA. Thermodynamic analysis showed that there was only one binding site while the interaction occurred. What’s more, ΔH > 0 and ΔS > 0 indicated that the hydrophobic force played a major role in the interaction. Docking results implied that both of the derivatives bound to the hydrophobic cavity of HSA by hydrophobic force and hydrogen bonding, wherein the hydrophobic force was the main force, which was consistent with the thermodynamic results.