Background: Atrial fibrosis is a landmark of cardiac remodeling to perpetuate atrial fibrillation (AF), and recent studies have indicated that microRNAs (miRNAs) are essential regulators of multiple cardiovascular disease processes. Herein, we aimed to investigate the relationship between circulating microRNA-21 (miR-21), atrial fibrosis, and AF in patients with atrial enlargement. Methods: A total of 60 persistent AF patients and 60 matched sinus rhythm (SR) controls were enrolled in the study. We measured their plasma miR-21 levels by using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Then, each patient underwent transthoracic echocardiography (TTE), while persistent AF patients underwent delayed enhancement magnetic resonance imaging (MRI). Results: The plasma miR-21 concentrations in the AF group were significantly higher than in the controls, and highly correlated [R=0.689, 95% confidence interval (CI): 0.527 to 0.802; P<0.001] with left atrial (LA) fibrosis measured by delayed enhancement MRI. Receiver operating characteristics (ROC) curve analysis showed that the area under the curve (AUC) of plasma miR-21 to identify AF was 0.813 (95% CI: 0.731 to 0.878). The increasing levels of circulating miR-21 were significantly associated with the higher risk of AF by using logistic regression analysis, even after adjustment for known confounding variables. Conclusions: Circulating miR-21 highly correlates with the quantification of LA fibrosis by using delayed enhancement MRI and is associated with the risk of persistent AF in patients with LA enlargement.