A novel label-free and PCR-free electrochemical method is demonstrated for simultaneous evaluation of double gene-specific methylation loci in a single-tube experiment without numerous hybridization and heating processes. The method composes three units: two electrochemical quantum dots tagged dual-functional hairpin probes (HP1-CdS and HP2-PbS) for methylation loci recognition, and capture probes modified magnetic beads (MB-CP) as isolation vehicle. A p53 gene fragment with two methylation loci was selected as a target model. After pretreatment with sodium bisulfite, samples were simply incubated with HP1-PbS, HP2-CdS, and MB-CP conjugates. The resultant MBs were then isolated and stripping analyzed by square wave voltammetry (SWV). From the SWV responses of the QDs barcodes hybridized on MBs, the methylation pattern and level of target gene fragment were clearly identified. The linear range was 5.0-125.0 nM with a limit of detection (LOD) of 13 nM for locus A which was tagged by PbS, and 0.5-125.0 nM with a LOD of 038 nM for locus B which was tagged by CdS. The method showed satisfactory accuracy and testing recovery. In comparison with the current methods, the proposed method is convenient and can evaluate two gene-specific methylation in a single-tube experiment within 2.5 h. With regard to the advantages of facility, low-cost, and ease of operation, the method can be a potential platform for identification of DNA methylation status in a simple way. (C) 2016 Elsevier B.V. All rights reserved.