Technological advances in the detection of circulating tumor DNA (ctDNA) have made new options available for diagnosis, classification, biological studies, and treatment selection. However, effective and practical methods for analyzing this emerging class of biomarkers are still lacking. In this work, a fluorescent biosensor was designed for the label-free detection of ctDNA (EGFR 19 del for non-small cell lung cancer, NSCLC). The biosensor was based on the fact that MnO2 nanosheets (MnO2 NSs) have stronger affinity towards single-stranded DNA (ssDNA), as compared with double-stranded DNA (dsDNA). As a high-performance nanoenzyme, MnO2 NSs could oxidize dopamine (DA) into fluorescent polydopamine nanoparticles (FL-PDA NPs), which could be used as a fluorescence signal. The probe ssDNA could be adsorbed on the surface of MnO2 NSs through π-π stacking, and the active site would be masked, causing a lower fluorescence. After the targets were recognized by probe ssDNA to form dsDNA, its affinity for MnO2 NSs decreased and the active site recovered, causing a restored fluorescence. It was verified that Mn ions, •OH radicals and electron transfer were the important factors in the catalytic oxidation of DA. Under the optimal experimental conditions, this biosensor exhibited a detection limit of 380 pM and a linear range of 25-125 nM, providing reliable readout in a short time (45 min). This sensor exhibited outstanding specificity, stability and reproducibility. In addition, this sensor was applied to the detection of ctDNA in serum samples and cell lysates. It is demonstrated that FL-PDA NPs can be used as a fluorescence signal for easy, rapid and label-free detection of ctDNA without any other amplification strategies, and the proposed strategy has great potential for biomarker detection in the field of liquid biopsy.