Tetracycline HCl is a new member of the class of antibiotics. Therefore, its effects on the secondary structural of Bovine Serum Albumin, and the mechanism of action are poorly understood. The interaction between Tetracycline HCL and BSA were investigated with fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, ultraviolet spectroscopy, circular dichroism (CD), infrared spectroscopy and molecular docking under imitated physiological conditions. The analysis of fluorescence spectra showed that Tetracycline HCl could strongly quenching the fluorescence of BSA and the quenching process is a static process. The effective binding constants (Ka) were calculated to be 2.813×105 L·mol-1（298 K） by stern-volmer equation. According to the Vant’s Hoff equation, the thermodynamic parameters were calculated to be ΔS=-151.1 J·mol-1·K-1 ， ΔH=-76.09 kJ·mol-1， indicating that the predominant forces in the complex were hydrogen bonding and Van der Waals forces. The results of synchronous fluorescence, UV spectrum , FT-IR , CD spectrum and three dimensional fluorescence spectrum, futher demonstrated that the secondary conformation and micro-environment of BSA has been changed after interaction with Tetracycline HCL. Based on the Fster’s theory of non-radiation energy transfer, and the specific binding distance between Tetracycline HCL-BSA system was 0.49 nm. Hill’s coefficients (nH<1) proved that a negative cooperativity was found when Tetracycline HCLbound to BSA. The alteration of the protein secondary structure were quantitatively calculated from circular dichrosim (CD) spectroscopy with increase of α-helix content about 9.16%(1∶1). The result of molecular docking simulation revealed that Tetracycline HCL was located in sudlow’s site I corresponding to subdomain IIA through multiple interactions-hydrogen bond, hydrophobic and vander waals, etc. This research will provide valuable information for understanding the action mechanism of Tetracycline HCl with BSA, and is helpful for understanding its effect on protein function during the storage and transport process.