N-doped hollow carbon spheres loaded with Cu single atoms (Cu SAs/N-CSs) were prepared via an in situ hardtemplate method. As an electrode catalyst, the Cu SAs/N-CSs electrochemical-sensing properties to hydroquinone (HQ) and catechol (CC) were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV), and the linear detection ranges of HQ and CC were 1−324 and 1−123 μM, respectively; the detection limits are 0.33 and 0.17 μM, respectively. The experimental and DFT calculation results show that Cu SAs/N-CSs have stronger adsorption strength and more effective affinity for CC and HQ due to the synergistic effect of dispersed Cu SAs and N-CSs, thus improving the electrocatalytic and sensing properties of CC and HQ. Furthermore, the prepared electrode has been successfully applied to the simultaneous determination of HQ and CC in practical water samples with satisfactory results.