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Nowadays, there are strong efforts in developing new technology for rapid detection of specific DNA sequences for environmental monitoring, forensic analysis and rapid biomedical diagnosis applications. That is where microfluidic paper-based analytical devices are positioned as suitable platforms for the development of point of care analytical devices, due to their simple fabrication protocols, ease of use and low cost. Herein, a methodology for in situ single strand DNA detection by using a colorimetric assay based on the formation of a DNAzyme within a paper substrate was developed. A DNAzyme that could only be formed in the presence of a specific sequence of the Y human amelogenin gene was designed. The performance of the DNAzyme was followed colorimetrically first in solution and then in paper substrates. The reaction was found to be specific to the Y fragment selected as analyte. The DNAzyme reaction on paper enabled the unequivocal colorimetric identification of the Y single strand DNA fragment both qualitatively, with the naked eye (143 ng), and quantitatively by image analysis (45.7 ng). As a proof of concept, a microfluidic paper-based device, pre-loaded with all DNAzyme reagents, was characterized and implemented for the simultaneous detection of X and Y single strand DNA fragments. This work was supported by Gobierno de España, Ministerio de Economía y Competitividad, with Grant No. BIO2016-80417-P; funding from Basque Government (Grant: IT1271-19) and European Union funds: DNASURF (H2020-MSCA-RISE-778001). E.A.-H. acknowledges funding from the Basque Government, Department of Education, for predoctoral fellowship 2016.