Nitazoxanide is one broad-spectrum antiparasitic drug, and its potential uses in clinic have attracted much attention in the past decades. The interaction of nitazoxanide with bovine serum albumin (BSA) was investigated systematically by fluorescence spectroscopy, UV–Vis absorption spectroscopy, CD spectroscopy and molecular docking study. The results indicated that the fluorescence quenching of BSA by nitazoxanide was static. The corresponding thermodynamic parameters ΔH, ΔS and ΔG calculated according to van’t Hoff equation revealed that both hydrophobic interaction and electrostatic interaction play great roles in the binding of nitazoxanide to BSA process CD spectroscopy which showed that the secondary structure of BSA is changed by nitazoxanide. Molecular docking study further indicated the strong binding affinity between nitazoxanide and BSA.