Cefazolin (CFZ) is the first generation of β-lactam semi-synthetic cephalosporin and which are mainly used to treat Gram positive and negative bacteria. Ceftriaxone (CRO) belongs to the third generation of β-lactam broad-spectrum antibiotic and are particularly useful in treating the disease caused by sensitive bacteria and post-surgery infection prevention. Human serum albumin (HSA) is the most abundant protein in plasma and can bind with a variety of endogenous and exogenous compounds reversibly, which is playing a role in storage and transport. Therefore, detailed investigating the interaction of CFZ and CRO with HSA are very important to understand the pharmacokinetic behavior of the CFZ and CRO. The interaction between CFZ and CRO with HSA were investigated with fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, circular dichroism (CD) and molecular docking and so on under imitated physiological conditions. The results showed that CFZ and CRO could interact with HSA to form complex at 298 and 310 K, which led to quench the intrinsic fluorescence of HSA via static quenching. After correcting the binding parameters, the Stern-Volmer quenching constants (KSV) and binding constants (Ka) of HSA-CFZ and HSA-CRO system inversely correlated with temperatures, and there was one binding site between HSA-CFZ or HSA-CRO system. Based on the Fster’s theory of non-radiation energy transfer, the specific binding distance between HSA-CFZ or HSA-CRO system was 2.41 or 1.40 nm, respectively. Hill’s coefficients (nH<1) proved that a negative cooperativity was found when CFZ or CRO bound to HSA. The thermodynamic parameters (ΔH(HSA-CFZ)=-22.67 kJ·mol-1, ΔH(HSA-CRO)=-39.56 kJ·mol-1, ΔS(HSA-CFZ)=-4.90 J·mol-1·K-1, ΔS(HSA-CRO)=-37.28 J·mol-1·K-1) demonstrated that CFZ and CRO can spontaneously bind with HSA through hydrogen bonds and van der Waals forces. Three-dimensional fluorescence spectroscopy and circular dichroism (CD) showed that CFZ and CRO could change the micro-environment and conformation of HSA. The results of molecular docking revealed that CFZ and CRO were located in Sudlow’s site Ⅰ of HSA proven bycompetitive binding experiments. This study will be helpful to understand the mechanism which affects the conformation and function of HSA with CFZ and CRO in biological processes.