Atrial fibrillation (AF) is explained by anatomical and electrophysiological changes in the atria determined by high pressure, dilatation, infiltration and inflammation in the myocardium. There are some biomarkers implicated in these processes, namely, NT-proBNP, high sensitivity troponin (Hs-Tn), urate, galectin-3, ST2, C reactive protein and fibrinogen. The aim of this study was to assess differences in these biomarkers between patients with AF and healthy controls. We designed a cross-sectional study consecutively including all patients undergoing electrical cardioversion in our hospital for persistent AF and matched healthy controls. We included 115 patients with persistent non-valvular AF and 33 healthy subjects. The biomarkers NT-proBNP, ST2 and Hs-Tn T were significantly related to the presence of AF (1054 ± 833.30 vs. 58.31 ± 59.40, p < 0.001; 35.43 ± 15.89 vs. 27.43 ± 10.95, p < 0.001 and 10.25 ± 6.11 vs. 8.42 ± 6.85, p < 0.001, respectively). NT-proBNP was the best biomarker differentiating AF patients (area under the curve 0.995). The best NT-proBNP cut-off point to differentiate AF was 102 pg/mL; for Hs-Tn T it was 11.5 ng/L and for ST2 it was 37.7 ng/mL. It is possible that these biomarkers intervene at the onset of AF and have no role in AF maintenance.