Dopamine (DA) and acetaminophen (AP) are involved in important physiological processes in vivo, while their detection in the complex biological matrix remains challenging. Permselective electrochemical sensors based on polydopamine composite films with an electro-reduced graphene oxide (rGO) were prepared by electrodeposition in a phosphate buffer solution of pH 7.4. Scanning electron microscopy, energy-dispersive X-ray analysis, and Raman spectroscopy were used to characterize the surface features of the modified electrodes. The electrochemical permselective behavior was investigated by electrochemical impedance spectroscopy, cyclic voltammetry, differential pulse voltammetry, and amperometric technique. Permselectivity of sensors to AP was evaluated by permeability and selectivity coefficient. With the layer-by-layer architecture, polydopamine exhibited high permeation to DA and AP but remained Fe(CN)6
3− inhibition. The depression to ascorbic acid (≥10 mM) was tested and the selective coefficient for uric acid even reached at −1.6. Based on excellent permselectivity, polydopamine/rGO composite sensors possessed low detection limit (0.2 and 0.45 μM for DA and AP, respectively), which made the specific method feasible for their simultaneous detection in urine and saliva samples. The long-term stability of composite films in biological samples was tested through amperometric monitoring for 12 h.