The binding affinity between hydroxyproline (Hyp) and human serum albumin (HSA) was investigated under simulated physiological conditions, using molecular modeling in combination with steady-state fluorescence, synchronous fluorescence, time-resolved fluorescence, UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Molecular modeling studies suggested that the Hyp molecule was situated within subdomain IIA of HSA. The fluorescence quenching analysis indicated that the fluorescence of HSA was quenched by Hyp with a dynamic quenching mechanism. The binding constants were calculated according to Scatchard's equation and implied that Hyp can bind to different binding sites on HSA. The thermodynamic analysis implied that hydrophobic forces were the main interaction in the Hyp–HSA system, which was found to be in line with the results of molecular modeling. Furthermore, the conformational structure of HSA was changed with various amounts of Hyp, which was confirmed by synchronous fluorescence, UV-vis absorption, CD, and FT-IR spectra.