Abstract: Acute myocardial infarction (AMI) ranks first among the death factors in China, and is the top priority in the clinical prevention and treatment of cardiovascular diseases. Myocardial troponin I (cTnI), as the most reliable clinical index of evaluating myocardial injury, is currently the preferred biomarker and gold standard for the diagnosis of myocardial infarction. Immunology method is a common method of clinical detection AMI, many domestic and foreign diagnostic reagent suppliers can produce cTnI detection kit, but immune testing system from different manufacturers will produce different value results for the same sample, on the one hand, the commercial kits of different suppliers in sensitivity, specificity and selectivity, on the other hand is different kits used in different antibodies, identify antigen epitope region, antibody and serum cTnI sample binding ability differences, resulting in inconsistent results between different manufacturers, thus affect the accuracy of clinical diagnosis. The consistency and accuracy of the quantitative results are closely related to the structure of cTnI. However, due to the complexity of cTnI, the structural characterization of cTnI in the solution state is very challenging. Through experimental condition optimization and data processing parameters, the characterization method of cTnI HDX, based on hydrogen deuterium exchange-mass spectrometry (HDX-MS) technology, is established, and applied to the study of cTnI. Meanwhile, this method has good universality and can be easily extended to cTnI conformation transition study and the development of kinetic method, which lays the technical foundation for promoting the standardization of cTnI from the perspective of protein structure.