Improving the detection of DNA hybridization is a critical issue for several challenging applications encountered in microarray and biosensor domains. Herein, it is demonstrated that hybridization between complementary single-stranded DNA (ssDNA) molecules loosely adsorbed on a mica surface can be achieved thanks to fine-tuning of the composition of the hybridization buffer. Single-molecule DNA hybridization occurs in only a few minutes upon encounters of freely diffusing complementary strands on the mica surface. Interestingly, the specific hybridization between complementary ssDNA is not altered in the presence of large amounts of nonrelated DNA. The detection of single-molecule DNA hybridization events is performed by measuring the contour length of DNA in atomic force microscopy images. Besides the advantage provided by facilitated diffusion, which promotes hybridization between probes and targets on mica, the present approach also allows the detection of single isolated DNA duplexes and thus requires a very low amount of both probe and target molecules.