An ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation was developed. In this assay, target DNA (tDNA) in solution was accumulated on a silanized substrate blocked with ethanolamine and bovine serum albumin (BSA) through a hybridization reaction between tDNA and capture DNA immobilized on the substrate. The tDNA on the substrate was labeled with quantum dots which had been modified with detection DNA and blocked with BSA. The fluorescence image of single QD-labeled tDNA molecules on the substrate was acquired using total internal reflection fluorescence microscopy. The tDNA was quantified by counting the bright dots on the image from the QDs. The limit of detection of the DNA assay was as low as 6.4 × 10(-18) mol L(-1). Due to the ultra-high sensitivity, the DNA assay was applied to measure the beta-2-microglobulin messenger RNA level in single human breast cancer cells without a need for PCR amplification.