Objective To investigate the clinical application of high-sensitivity cardiac troponin T (hs-cTnT) in the diagnosis of patients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS). Methods The clinical data of 126 patients with NSTE-ACS admitted to the Department of Emergency of the Third People's Hospital in Yunnan Province from July 2016 to June 2017 were retrospectively analyzed, including 76 patients in non-ST-segment elevation myocardial infarction (NSTEMI) group and 50 patients in unstable angina (UA) group. Moreover, the clinical data of venous blood hs-cTnT and creatine kinase MB (CK-MB) were collected at immediate admission, 4 hours and 12 hours after admission to the hospital in the two groups. At last, the differences in levels of hs-cTnT and CK-MB were compared between the two groups. Results The hs-cTnT and CK-MB in NSTEMI group were gradually increased with the prolongation of therapeutic time, the elevation ratios of hs-cTnT were 88.2% (67 cases) and 100.0% (76 cases), 100.0% (76 cases) at admission and 4 hours, 12 hours after admission respectively; and the elevation ratios of CK-MB were 60.0% (30 cases), 80% (40 cases) and 96% (48 cases) respectively at the above time points; there were no significant differences in UA group. The levels of hs-cTnT and CK-MB in NSTEMI group were significantly higher compared to those of UA group at admission and 4 hours, 12 hours after admission [hs-cTnT (μg/L): the levels were respectively 0.182±0.052 vs. 0.010±0.001, 2.421±0.084 vs. 0.011±0.012, 5.647±0.012 vs. 0.11±0.012, CK-MB (U/L): the levels were respectively 36.3±12.1 vs. 8.1±0.5, 179.6±26.3 vs. 8.5±0.5, 286.0±23.2 vs. 7.9±0.7, all P ＜ 0.05]. Conclusion The blood level of hs-cTnT in acute myocardial infarction (AMI) patients is obviously increased, which can be used as the basis to distinguish NSTEMI from UA.