Acute coronary syndrome (ACS) patients treated with dual antiplatelet therapy (DAPT) show individual differences in platelet reactivity (PR). Here, we aim to find differences in the platelet microRNA (miRNA) expression profiles of high PR and low PR patients to serve as potential biomarkers. ADP-induced platelet aggregation (PAG) was used to determine PR. High-throughput sequencing technology was used to profile differentially expressed platelet miRNAs in high PR (PAG>50%) and low PR (PAG≤50%) patients. We used real-time quantitative reverse transcription-polymerase chain reactions (RT-qPCR) to validate the sequencing results. Finally, we statistically evaluated the diagnostic value of the miRNAs and explored their molecular function using bioinformatic analysis. The results show that miR-204-5p was confirmed to be significantly upregulated in the high PR group. The area under the ROC curve (AUC) of miR-204-5p was 0.667, and its expression significantly correlated with the Gensini score. Stepwise binary logistic regression analysis suggested that miR-204-5p expression level was an independent predictor of PR. Furthermore, bioinformatic analysis showed that miR-204-5p may be associated with platelet synapse formation and platelet vesicle release. Our data indicates that platelet miR-204-5p may serve as a novel biomarker of PR to guide treatment with antiplatelet drugs. © 2019 by the Association of Clinical Scientists, Inc.