Fluorescent aptamer probes physisorbed on graphene oxide (GO) have recently emerged as a useful sensing platform. A signal is generated by analyte-induced probe desorption. To address nonspecific probe displacement and the false positive signal, we herein report a covalently linked aptamer probe for adenosine triphosphate (ATP) detection. A fluorophore and amino dual modified aptamer was linked to the carboxyl group on GO with a coupling efficiency of ∼50%. The linearity, specificity, stability, and regeneration of the covalent sensor were systematically studied and compared to the physisorbed probe. Both sensors have similar sensitivity, but the covalent one is more resistant to nonspecific probe displacement by proteins. The covalent sensor has a dynamic range from 0.125 to 2 mM ATP in buffer at room temperature and is resistance to DNase I. Intracellular ATP imaging was demonstrated using the covalent sensor, which generated a higher fluorescence signal than the physisorbed sensor. After the cells were stimulated with 5 mM Ca(2+) for ATP production, the intracellular signal enhanced by 31.8%. This work highlights the advantages of covalent aptamer sensors using GO as both a quencher and a delivery vehicle for intracellular metabolite detection.