Background: In-stent restenosis (ISR) usually develops from stent neointimal hyperplasia (SNH), which will seriously weaken the effect of treatment. In this study, both SNH and ISR were classified as in-stent stenosis (ISS), and a non-invasive parameter – myocardial blood flow index (MBFI) was used to analyze its value in the diagnosis of stent abnormalities. Objectives: Analyzing the application of MBFI in the diagnosis of ISS. Patients and Methods: Clinical follow-up data for 572 patients with drug-eluting stent (DES) was collected continuously. Ninety cases were screened with the protocol of computed coronary tomography angiography (CCTA) and invasive coronary angiography (ICA) for the unrelieved symptoms. In-stent abnormalities included SNH (ISS < 50%) and ISR (ISS ≥ 50%). The ROC curve was analyzed using the optimal cutoff value of MBFI to evaluate the in-stent abnormalities. T-test of independent samples was used for the comparison data with normal distribution, and chi-square test was used for comparison of nominal variables P < 0.05 was considered statistically significant. Results: Frequency of ISS was not statistically different between genders (χ2 = 0.105) (P = 0.7463). The optimal cutoff value was 0.082 with the area under the curve (AUC) of 0.829 (P < 0.001). The sensitivity, specificity, positive predictive value, and negative predictive value were 91.4%, 89.1%, 84.2%, and 94.2%, respectively, and the accuracy was 90.0%. Among 39 cases with MBFI ≤ 0.082, 34 (87.18%, 34/39) were with ISS, including 18 (20%, 18/90) of SNH, and 16 (17.9%, 16/90) of ISR. In the 39 cases with MBFI ≤ 0.082, there were eight (20.5%, 8/39) presented new lesions, and seven (17.9%, 7/39) presented severe lesions. Conclusion: MBFI could be used for evaluating ISS, and more attention should be paid to the new accompanying lesions for the high risk of severe lesions.