An electrochemical sensor for the quantification of o-nitrophenol (o-NP) has been developed based on the β-cyclodextrin functionalized graphene nanosheets modified glassy carbon electrode (CD-GNs/GCE). The results indicated that CD-GNs showed good electrochemical behavior to the redox of o-NP which is attributed to the combination of the excellent properties of graphene and cyclodextrin. The peak currents possess a linear relationship with the concentration of o-NP in the range of 5–400 μM. The detection limit of o-NP reached to 0.3 μM on the basis of the signal-to-noise characteristics ( ). The peak potentials for the reversible redox waves are not affected by other nitrophenol isomers (m, p-NP), illustrating good selectivity. Furthermore, the developed electrochemical sensor exhibited good stability and reproducibility for the detection of o-NP and could be used to determine o-NP in real water sample.