Objective To study the relationship between serum levels of homocysteine (HCY), creatine kinase isoenzyme (CK-MB) and cardiac troponin I (cTnI) and severity of vascular stenosis, prognosis in patients with coronary artery disease. Methods 125 patients with coronary heart disease (CHD) and 100 healthy examiners enrolled in our hospital from January 2017 to January 2018 were included in the study. The differences of serum levels of HCY, CK-MB and cTnI were compared between CHD patients and healthy control group. The levels of serum HCY, CK-MB and cTnI were compared among patients with different lesion types, different stenosis counts, different Gensini scores and different prognosis. The relationship between serum levels of HCY, CK-MB and cTnI and severity of vascular stenosis and clinical prognosis in patients with CHD were observed. Results The serum levels of HCY, CK-MB and cTnI in healthy control group were significantly lower than those in CHD patients (P＜0.05). The concentrations of serum HCY, CK-MB and cTnI were gradually increased in SAP group, UAP group and AMI group (P＜0.05). They were gradually increased in single-stenosis group, double-stenosis group and multiple stenosis groups (P＜0.05). They were gradually increased in low Gensini group, middle Gensini group and high Gensini group (P＜0.05). Pearson correlation analysis showed that serum HCY, CKMB and cTCnI were positively correlated with coronary lesions counts (r=0.641, 0.789, 0.912, all P＜0.05), and was also positively correlated with Gensini score (r=0.5671, 0.712, 0.813, all P＜0.05). Follow up for 1 months, the serum levels of HCY, CK-MB and cTnI in patients with ACS in poor prognosis group were significantly higher than those in good prognosis group (P＜0.05). Conclusion The serum levels of HCY, CK-MB and cTnI have good application value in evaluating the lesion severity and the prognosis of patients with coronary artery disease.