Because of its rich π electrons, graphene oxide (GO) can bind with single-stranded DNA (ssDNA) through π-π stacking, but it cannot bind with double-stranded DNA (dsDNA). This different binding ability of GO with DNA has found many applications. By combining this ability with aptamer chemistry, we have developed a highly selective method for the detection of adenosine triphosphate (ATP). The hybrid of ATP aptamer with fluorescein (FAM)-labelled complementary DNA (FAM-DNA) demonstrates a weak affinity for GO and strong fluorescence of FAM-DNA. If ATP is present the strong fluorescence of FAM-DNA is quenched, because the binding of ATP with its aptamer greatly inhibits hybridization of the aptamer with FAM-DNA, and the unhybridized FAM-DNA is adsorbed on to the surface of GO. The quenched fluorescence intensity was found to be in proportion to the concentration of ATP in the range 3–320 μM with a detection limit of 0.45 μM. This method of ATP detection is highly selective and the existence of GTP, CTP and UTP have scarcely effect the determination.