Many cephalosporins are widely used, and the structures have specific shared characteristics. However, there is still a lack of systematic comparative research on the property differences between different structures. With the continuous development of research technology, the combination of spectroscopic technology and quantum chemical calculation has become one of the most effective methods to explore structural behavior and observe the electronic structure of drug molecules. This work aimed to systematically and comprehensively analyze the relationship between the structure and properties of non-prodrug-type third-generation cephalosporins.In this study, the density functional theory method X3LYP/6-311+G(d, p) was applied to conduct in-depth research on its geometric structure, infrared spectrum, ultraviolet spectrum, frontier molecular orbital, molecular electrostatic potential, reactivity descriptors, and further molecular docking. The theoretical values including ceftizoxime (CZX), were consistent with the crystal structures. The basic structure of cephalosporin (BSC) is similar in dihedral angle and bond angle, but atoms such as N and O greatly influence the bond length. The IR spectra of ceftazidime (CZX) and cefotaxime (CTX) calculated in theory agree well with the measured values in the experiment. The CO stretching vibration of the non-prodrug-type third-generation cephalosporin carbonyl group is mainly concentrated between 1 766 and 1 644 cm-1. The strong peaks in the range of 1 650~1 550 cm-1 are caused primarily by N—H in-plane bending vibration, and the primary amines will produce strong peaks in the range of 1 340~1 020 cm-1. The most substantial absorption peaks in the UV-Vis spectrum are mainly concentrated in the vicinity of 200~250 and 300~350 nm, and electron orbital transitions, including HOMO, contribute their main components to LUMO+1, HOMO-2 to LUMO, and HOMO-1 to LUMO+2. Molecular surface electrostatic potential and local reactivity descriptors analysis predict that the maxima and minima are mainly distributed near the sites where hydrogen atoms, carbonyl O atoms, and N atoms of amino groups are concentrated, respectively. Frontier molecular orbitals gap and global reactivity descriptors showed that ceftazidime(CAZ) is the most active molecule, but cefoperazone(CFP) is the opposite. A molecular docking study showed that the interaction with HSA was dominated by hydrophobic interaction, hydrogen bond, and π-cation interaction, and the results of the electrostatic potential analysis were verified. The spectral information and structural properties of non-prodrug-type third-generation cephalosporins obtained in this study based on density functional theory can provide vital information for quality control in preparing imitation products, new drug screening and development of derivatives, and understanding and analyzing mechanisms of action.