To investigate the expression and clinical significance of F0 ATP synthase C subunit (Csub) in patients with ischemic heart disease (IHD). The 101 patients with chest pain admitted to the department of emergency of the People's Hospital of Yuhuan from May 2019 to December 2020 were enrolled, including 59 patients with acute myocardial infarction (AMI) and 42 patients with unstable angina pectoris (UAP). At the same time, 50 age-matched healthy subjects in the health examination center were selected as the healthy control (HC). All patients had completed blood sampling before the intervention of drugs or other intervention measures in the emergency room. The content of serum Csub was detected by enzyme linked immunosorbent assay (ELISA), and the relationship between Csub and clinical characteristics was analyzed. At the same time, the contents of hypersensitivity cardiac troponin T (hs-cTnT) and MB isoenzyme of creatine kinase (CK-MB) in blood were detected by electrochemical luminescence. The receiver operator characteristic curve (ROC curve) was drawn to evaluate the value of Csub, hs-cTnT, and CK-MB in the early diagnosis of IHD. The baseline data such as age, gender, and history of the three groups were balanced. There were significant differences in low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), CK-MB, hs-cTnT and B-type natriuretic peptide (BNP), but there were no significant differences in other biochemical indexes. The Csub content in the AMI group and the UAP group were significantly higher than those in the HC group [8.96% (6.37%, 11.53%), 4.27% (3.23%, 6.49%) vs. 1.56% (1.07%, 2.33%), both P < 0.01]. Moreover, the Csub in the AMI group with more severe myocardial ischemia was higher than UAP group [8.96% (6.37%, 11.53%) vs. 4.27% (3.23%, 6.49%), P < 0.01]. A total of 59 patients with AMI were treated with percutaneous coronary intervention (PCI). According to the median of Csub, AMI patients were subdivided into above-median group (29 cases) and below-median group (30 cases). The results showed that there were no significant differences in the number of coronary artery lesion branches, the number of stent implantation and postoperative medication between the two groups. ROC curve analysis showed that the area under the curve (AUC) and 95% confidence interval (95%CI) of Csub, hs-cTnT and CK-MB in the diagnosis of IHD were 0.98 (0.95-1.00), 0.99 (0.99-1.00), 0.94 (0.89-0.99), respectively. The diagnostic efficacy of Csub was slightly lower than that of hs-cTnT but higher than that of CK-MB. When the cut-off value of Csub was 4.74%, the sensitivity and specificity for the diagnosis of IHD were 100% and 87.0%, respectively. Csub increased significantly in the serum of IHD patients, and further increased with the severity of ischemia. It can be used as a new diagnostic biomarker for the diagnosis and evaluation of the development of myocardial ischemia.