Background The aim of this study was to investigate the value of electrocardiograms (ECGs) and serological examinations in the differential diagnosis of acute pulmonary embolism (APE) and acute non‐ST elevation myocardial infarction (NSTEMI) in order to reduce the rate of clinical misdiagnosis. Methods The clinical data of 37 patients with APE and 103 patients with NSTEMI admitted to our hospital were retrospectively analyzed. The differences in the clinical manifestations, ECGs, myocardial zymograms, D‐dimers, and troponin (cTn) of the two groups were compared. Results In the patients with APE, the main symptom—found in 25 cases (67.56%)—was dyspnea, while in the patients with NSTEMI, the main symptom—found in 52 cases (50.49%)—was chest tightness. The incidences of sinus tachycardia and SIQIIITIII in the group of patients with APE were higher than in the group of patients with NSTEMI, and the difference was statistically significant (p < .05). There was no statistical significance in the difference of aspartate aminotransferase and lactate dehydrogenase (LDH) in the two groups (p > .05), although there was a statistically significant difference of creatine kinase (CK) and the creatine kinase isoenzyme‐MB (CK‐MB) in the two groups (p < .05). The levels of D‐dimers and cTn were increased in both groups, but the level of D‐dimers in the group of patients with APE was higher than that in the group of patients with NSTEMI. Conclusion With the occurrence of clinical manifestations like dyspnea, chest tightness, chest pain, and palpitation of unknown causes, the possibility of APE and NSTEMI should be considered.