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A turn-on fluorometric method is described for the determination of adenosine-5′-triphosphate (ATP). It is based on the displacement of a dye-labeled oligonucleotide from a cobalt(II) based layered double hydroxide (LDH). Due to the electrostatic and ligand exchange interaction, the FAM-labeled DNA is readily adsorbed on the LDH. This leads to complete and fast quenching of the green fluorescence of the label. However, on addition of ATP, the DNA is detached from the LDH because of the stronger affinity of ATP for LDH. This results in the restoration of the green fluorescence. The effect was used to design a sensitive assay that has a linear response in the 0.5–100 μM ATP concentration range and a 0.23 μM lower detection limit. It was applied to the determination of ATP in spiked serum samples. Graphical abstract Schematic presentation of a fluorometric ATP assay based on the displacement of a dye-labeled oligonucleotide from a layered double hydroxide (LDH). [ABSTRACT FROM AUTHOR]