Contamination of antibiotic residues in food and water is an understudied safety concern and is increasingly gaining importance worldwide due to the rising rates of drug resistant organisms. As a class of antibiotics targeting β-lactam, cephalosporin is frequently used in treatment of bacterial infections. In this study, molecular modeling approaches in combination with absorbance, fluorescence and circular dichroism spectroscopy was employed to investigate the interactions of four cephalosporins (cefalexin, cefaclor, cefixime and cefepime) with human serum albumin (HSA). In the mechanism discussion, it was proved that the fluorescence quenching of HSA by cephalosporin is a result of the formation of cephalosporin-HSA complex. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that cephalosporin bind to HSA with the binding affinities of the order 103–104L·mol−1. The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and negative entropy changes and the hydrogen bonds and van der Waals interactions may play major role in complex formation. Furthermore, the investigated result indicates that azyl served as a hydrophilic group to change the chemical property for drug, the chlorine atom on the R1-group of cefaclor weakened the overall molecular polarity or hydrophilicity, while the binding site on HSA was determined by the carboxylate of cefepime. Some other structural characteristics for four kind of cephalosporins were also exhibited in this paper.