In recent years, numerous aptamers have been physisorbed on graphene oxide (GO) to develop fluorescence resonance energy transfer-based aptasensors using the fluorescence quenching property of GO. However, physisorbed aptasensors show poor signal reversibility and reproducibility as well as nonspecific probe displacement, and thereby are not suitable for many analytical applications. To overcome these problems when working with complex biological samples, we developed a facile and robust covalent surface functionalization technique for GO-based fluorescent aptasensors using a well-studied adenosine triphosphate binding aptamer (ABA). In the scheme, GO is first modified with amino-silane, and further with glutaraldehyde to create available carbonyl groups for the covalent attachment of a fluorophore and an amino dual modified ABA. The surface modification method was characterized by zeta-potential, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy (FTIR). The linearity, sensitivity, selectivity, and reversibility of the resulting GO-based covalent aptasensor was determined and systematically compared with the physisorbed aptasensor. Although both sensors showed similar performance in terms of sensitivity and linearity, better selectivity and higher resistance to nonspecific probe displacement was achieved with the developed covalent ABA sensor. The surface modification technique developed here is independent of the aptamer sequence, and therefore could be used universally for different analytical applications simply by changing the aptamer sequence for the target biomolecule.