Minocycline (MC) is a semisynthetic tetracycline broad-spectrum antibiotic with stronger antibacterial activity, which is absorbed rapidly after oral administration, and the binding rate to serum protein ranges from 76% to 83%. The study of the binding mechanism between Bovine serum albumin (BSA) and MC is helpful to explore the interaction mechanism between MC and BSA at the molecular level, to further understand the structure and functional relationship of BSA and MC, and to provide necessary data support for the further study of pharmacological toxicity and efficacy of MC. The interaction between MC and BSA has been investigated by fluorescence spectroscopy, circular dichroism, ultraviolet spectroscopy and molecular docking simulation at different temperature and simulated physiological conditions. The results show that MC quenches the fluorescence of BSA, and the quenching constant decreases with the increase of temperature. This indicates that the quenching mechanism of MC and BSA is static quenching. The fluorescence results were calculated using the Stern-Volmer equation and the static quenching double logarithmic formula, and the results showed that the number of binding sites n between MC and BSA is close to 1. According to Van’t Hoff thermodynamic equation at 298, 303 and 308 K, the thermodynamic parameters were obtained as follows: enthalpy change ΔH=-34.14 kJ·mol-1, entropy change ΔS=32.55 J·(mol·K)-1, Gibbs free energy ΔG=-43.84 kJ·mol-1 (298 K), -43.88 kJ·mol-1 (303 K), -44.17 kJ·mol-1 (308 K), which proved that the main force between MC and BSA is the van der Waals and hydrogen bonding, and the process of its action is the spontaneous and exothermic process. Through the UV-visible absorption spectrum analysis of BSA and MC, the position of the absorption peak of BSA has a significant red shift, indicating that the conformation of BSA has changed. According to Frster’s theory of non-radiative energy transfer, the binding distance r between MC and BSA is 1.873 nm, which indicates that non-radiative energy transfer occurs between MC and BSA. In addition, the experimental results of synchronous fluorescence spectroscopy showed that the conformation of BSA changed when MC interacted with BSA, and the binding site was on tryptophan (Trp) residues. The results also showed that the conformation of BSA changed by three-dimensional fluorescence spectroscopy and circular dichroism, and (Trp) the polarity of the surrounding environment decreased and hydrophobicity increased. Quantitative analysis of secondary structure of circular dichroism before and after the interaction of MC and BSA showed that the content of alpha-helix structure in BSA was 31.75%. After adding MC gradually, the content of α-helix structure changed to 47.10% (cBSA∶cTRO=1∶1) and 54.39% (cBSA∶cTRO=1∶5), indicating that the content of α-helix structure increased, and the structure of BSA was mainly α-helix structure. Molecular docking simulation showed that MC interacts into the site I (subdomain IIA) of BSA, it forms van der Waals interaction between MC and the amino acid residues PHE508, LYS535, HIS534, PHE501, GLN579, VAL546, MET547, LEU528, PHE508 of BSA, hydrogen bonds formed between MC and the amino acid residues LYS524 and LEU531, and super conjugation also formed between MC and the amino acid residues ALA527, VAL575, LEU531, PHE508. These amino acids bind closely with MC molecules, and MC changes the secondary structure of BSA. The data obtained in this experiment are helpful to understand the interaction mechanism between MC and BSA, as well as the effect of MC on BSA conformation during storage and transportation.