Objective To prepare mouse-derived monoclonal antibody against human suppression of tumorigenicity 2 (ST2) molecule and make initial identification. Methods BALB/c mice were immunized with the recombinant human ST2 molecule, and the conventional B-cell hybridoma technology was used to prepare the anti-ST2 monoclonal antibodies (mAbs). Their application in western blotting, immunohistochemistry, and flow cytometry were evaluated. The sandwich ELISA detecting soluble ST2 was established to test the serum levels of ST2 in patients with heat stroke. And the ST2 luciferase reporter gene detection system was established to detect their neutralization activity. Results Thirty-eight hybridoma cell lines secreting mouse anti-human ST2 mAb were obtained and named from XA325.1 to XA325.38. Preliminary screening and identification of them showed that they can be used to identify the purified recombinant ST2 proteins and cellular expressed ST2 using western blotting and immunohistochemistry. Two of them can be used for flow cytometry to identify the exogenously transfected ST2 molecule on the cell surface. Using XA325.16 mAb coating, combined with XA325.5-labeled biotin, an ELISA kit detecting soluble ST2 in serum was established. It was found that the serum levels of ST2 in patients with heat stroke increased significantly. Moreover, XA325.5 was found with neutralizing activity which can block the biological effect of IL-33. Conclusion A set of mouse anti-human ST2 mAbs was prepared, which can be used in a variety of immunological detection techniques. Besides, XA325.5 neutralizing antibody has a potential value in clinical application.