•This novel strategy is based on ligand-responsive G-quadruplex formation.•The assay is label free for rapid DNA detection.•This probe is simple and cost-efficiency in design and operation.A facile and label-free assay with label-free molecular beacons (MBs) and fluorescent dye N-methyl mesoporphyrin IX (NMM) was developed for the detection of specific single-stranded DNA sequences. It was demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 20 bases) of RNA fragment of human immunodeficiency virus (HIV) as model systems. In the absence of target DNA, the MBs were in the stem-closed form, the G-quadruplex structure could not form and the fluorescence signal of NMM was very low. In the presence of target DNA the MBs turned “Off” to “On”, thus promoting the formation of G-quadruplex which could greatly enhance the fluorescence of NMM. This biosensor was simple in design, fast in operation, and more convenient and promising than other methods. It took less than 30 min to finish and its detection limit was 1.4 nM. No sophisticated experimental techniques or chemical modification for DNA sequences were required. This new approach could be widely applied to sensitive and selective nucleic acids detection.