Detection of specific DNA sequences is critical in life science. In this study, we investigated the interaction of cobalt oxyhydroxide (CoOOH) nanoflakes with DNA and their fluorescence quenching mechanism of a FAM-labeled single-stranded DNA (ssDNA) probe. ssDNA could adsorb on the CoOOH surface via electrostatic interactions and therefore the fluorescence of FAM was quenched. However, upon addition of targets, ssDNA was hybridized with target DNA and the formed double-stranded DNA (dsDNA) had much weaker affinity to CoOOH, resulting in the retaining of fluorescence. Based on the affinity difference of CoOOH nanoflakes to ssDNA and dsDNA and fluorescence resonance energy transfer based fluorescence quenching, a mix-and-detect method was proposed for homogeneous detection of DNA. The proposed method is simple and can be finished in a few minutes with high sensitivity. Furthermore, it displays a wide linear range from 1 to 50 nM with a detection limit of 0.5 nM and is capable of detecting DNA in real biological samples.