Aim To analyze the changes of serum microRNA-224 (miR-224) level after percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS) and its relationship with in-stent restenosis. Methods A total of 212 patients with ACS who were treated from January 2016 to December 2018 were selected as study subjects. They were divided into two groups based on the occurrence of in-stent restenosis:stenosis group (n=42) and non-stenosis group (n=170). The serum miR-224 level of ACS patients was detected by real-time quantitative PCR, and the relationship between miR-224 level and in-stent restenosis was analyzed. Results In 212 patients with ACS who underwent PCI treatment, 42 patients (19.81%) developed in-stent restenosis within 1 year. The low density lipoprotein cholesterol (LDLC), the proportion of diabetes and the length of the stent were higher in the stenosis group than those in the non-stenosis group, and the differences were statistically significant (P＜0.05). The level of serum miR-224 in the two groups showed an increasing trend with time. The 7-day miR-224 level was lower in the stenosis group than that in the non-stenosis group, and the difference was statistically significant (P＜0.05). The 7-day miR-224 assessment of in-stent restenosis in patients with ACS was 0.860, which was higher than the 1-day miR-224 and the 3-day miR-224 (P＜0.05).Logistic multivariate regression analysis showed that LDLC, diabetes, stent length, and 7-day miR-224 level were closely related to in-stent restenosis in ACS patients (P＜0.05). Conclusion The level of miR-224 is closely related to in-stent restenosis at 1 year after PCI in patients with ACS. A low serum miR-224 level at 7 days after PCI in patients with ACS indicates a higher risk of in-stent restenosis.