Acute myocardial infarction (AMI) is a cardiovascular disease with high morbidity and mortality, and microRNA-139-5p (miR-139-5p) has been reported to be closely related with myocardial viability. This study aimed to investigate the effects of miR-139-5p on vascular endothelial cells, detect miR-139-5p expression in AMI patients and evaluate its diagnostic value. A dual-luciferase reporter assay was utilized to confirm the interaction of miR-139-5p with vascular endothelial growth factor receptor-1 (VEGFR-1). Quantitative real-time PCR was used to detect the levels of miR-139-5p and VEGFR-1 in serum and cells. The viability of human umbilical vein endothelial cells (HUVECs) was measured using a cell counting kit-8 assay. The correlation between miR-139-5p and VEGFR-1 was analyzed by Pearson correlation analysis. The diagnostic value of miR-139-5p, cardiac troponin I (cTnI) and creatine kinase isoenzymes (CK-MB) was identified by receiver operating characteristic analysis. miR-139-5p suppressed cell viability by directly targeting VEGFR-1 in HUVECs. Increased miR-139-5p and decreased VEGFR-1 levels were found in AMI patients and hypoxia-treated HUVECs, and miR-139-5p and VEGFR-1 were shown to be negatively correlated. The diagnostic value of miR-139-5p for AMI screening was high, and the combination of cTnI, CK-MB and miR-139-5p had the highest diagnostic accuracy. miR-139-5p inhibited cell viability by inhibiting VEGFR-1 in hypoxia-treated HUVECs. miR-139-5p inhibits endothelial cell viability of AMI by inhibiting VEGFR-1, and increased miR-139-5p expression in AMI patients has high diagnostic value for AMI screening, indicating that miR-139-5p may serve as a diagnostic biomarker and molecular therapeutic target for AMI.