Abstract: Alternaria mycotoxins, as a secondary metabolite produced by Alternaria species, widely contaminates fruits, vegetables and food crops and often enters the human food chain through multiple channels, causing food safety issues. In order to study the absorption, distribution, transport and metabolism of tenuazonic acid (TeA) in animals, steady state fluorescence spectrum, synchronous fluorescence spectrum, 3D fluorescence spectrum and circular dichrochromatic spectroscopy were used to determine the molecular interaction and mechanism between TeA and human serum albumin (HSA) and orbovine serum albumin (BSA). Results showed that: TeA displayed a static quenching effect on both types of serum albumin under the simulated blood physiological pH of 7.40 to form a stable non-fluorescent complex, the number of binding sites indicated that TeA and serum albumin combined data ratio of 1:1. Thermodynamic analysis demonstrated that the forces involved in the interaction between TeA and two serum albumins were mainly hydrophobic forces (ΔH>0, ΔS>0). 3D fluorescence and synchronous fluorescence results suggested that the combination of TeA changed the microenvironment polarity of the amino acid residues of serum albumin. Circular dichroism showed that the combination of TeA and serum albumin increased the content of α-helical structure in HSA and BSA. The binding of TeA to HAS and BSA could occur spontaneously and had a strong binding capacity to change the conformation of both serum albumin after binding. The results would lay a solid foundation for further studies such as exposure and metabolic dynamics of streptospore toxin in animals.