The biological application of thiosemicarbazide transition metal complexes is the focus of current research. In order to better understand the interaction mechanism between thiosemicarbazide aryl ruthenium complexes and has, two thiosemicarbazide aryl ruthenium(Ⅱ) complexes were synthesized. The fluorescence quenching mechanism of two thiosemicarbazide aryl ruthenium complexes with human albumin was studied by time-resolved fluorescence spectroscopy and steady-state fluorescence spectroscopy. Fluorescence spectroscopy results indicate that these two thiosemicarbazide aryl ruthenium complexes could quench the endogenous fluorescence of has, and the fluorescence quenching effect of HSA was linear with the concentration of the complex. It was found that the fluorescence quenching efficiency of complex 2 was stronger. The quenching and binding constants of the two complexes interacting with HSA decrease with the increase in temperature. Therefore, the interaction process between the complexes and HSA was a static quenching process, and the fluorescence quenching ability of complex 2 was stronger. Through the analysis of thermodynamic parameters, the main binding forces between the two complexes and HSA are hydrogen bond, and van der Waals force, and the binding process between the two complexes and HSA was spontaneous. Finally, the infrared absorption and circular dichroism spectra show that the two thiosemicarbazide aryl ruthenium complexes have different effects on the secondary conformation and microenvironment of HSA. The infrared absorption spectra show that combining the two complexes with HSA causes the rearrangement of the secondary structure of HSA, Circular dichroism spectra showed that the addition of these two complexes reduced the secondary structure stability of HSA. The above studies show that exploring the effect of thiosemicarbazide aryl ruthenium complex on the structure and function of human serum albumin can reveal its possible mechanism of action with HSA after entering the body as an antitumor drug. Therefore, it provides a theoretical reference for researching and developing aryl ruthenium complex anti-tumor drugs with thiosemicarbazide as the ligand.