Simultaneous electroanalysis of isoniazid and uric acid (UA) was achieved at a poly(sulfosalicylic acid) (PSA) and carboxylated graphene (CG)-modified glassy carbon electrode (GCE). The CG cast-coated on the GCE was cathodically reduced to form electroreduced CG (ERCG), while the PSA was electrochemically synthesized on the ERCG/GCE. Electrochemical quartz crystal microbalance was used to investigate the electrodeposition processes. The surface morphologies and electrochemical properties of the PSA/ERCG/GCE were studied by scanning electron microscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy. The electrochemical behaviors of isoniazid and UA at PSA/ERCG/GCE were investigated by CV and differential pulse voltammetry (DPV), giving well defined and well separated oxidation peaks of isoniazid and UA. The PSA/ERCG/GCE exhibited notably enhanced electro-oxidation signals of isoniazid and UA in NH3-NH4Cl buffer solution (pH 9.0), as compared with bare GCE and PSA/GCE. Under optimum conditions, the DPV peak currents at PSA/ERCG/GCE responded linearly to isoniazid concentration from 0.05 to 15μM and to UA concentration from 0.02 to 15μM, with limits of detection of 12nM for both isoniazid and UA, which is an improvement compared to many other reported techniques. The PSA/ERCG/GCE was successfully applied to the simultaneous determination of isoniazid and UA in urine substrate samples.