A nanocomplex was developed for molecular sensing in living cells, based on the fluorophore-labeled aptamer and the polydopamine nanospheres (PDANS). Due to the interaction between ssDNA and PDANS, the aptamer was adsorbed onto the surface of PDANS forming the aptamer/PDANS nanocomplex, and the fluorescence was quenched by PDANS through Förster resonance energy transfer (FRET). In vitro assay, the introduction of adenosine triphosphate (ATP) led to the dissociation of the aptamer from the PDANS and the recovery of the fluorescence. The retained fluorescence of the nanocomplex was found to be linear with the concentration of ATP in the range of 0.01-2 mM, and the nanocomplex was highly selective toward ATP. For the strong protecting capability to nucleic acids from enzymatic cleavage and the excellent biocompatibility of PDANS, the nanocomplex was transported into cells and successfully realized "signal on" sensing of ATP in living cells; moreover, the nanocomplex could be employed for ATP semiquantification. This design provides a strategy to develop biosensors based on the polydopamine nanomaterials for intracellular molecules analysis. For the advantages of polydopamine, it would be an excellent candidate for many biological applications, such as gene and drug delivery, intracellular imaging, and in vivo monitoring.