Caffeine (CF) is a member of the methylxanthine family with numerous biological activities, which may contribute to the prevention of human disease but also may be potentially harmful. In the present study, the interaction of CF with bovine hemoglobin (BHb) under physiological condition was studied by fluorescence and UV/vis spectroscopy. Fluorescence data revealed that the fluorescence quenching of BHb by CF was the result of the formed complex of CF-BHb. The binding constants and thermodynamic parameters at three different temperatures, the binding position, and the binding force were determined. The hydrophobic and hydrogen bonds interactions were the predominant intermolecular forces to stabilize the complex. The conformation of BHb was discussed by synchronous fluorescence techniques. The synchronous spectra indicated that the structures of the Tyr and Try residues environments were altered and the physiological functions of BHb were affected by 0. This study provides important insight into the mechanism of erythrocyte sickling, which may be a useful guideline for further toxicology investigation.