In this work, an electroanalytical evaluation for voltammetric sensing of the anti-rheumatic sulfasalazine (SSZ) at an unmodified screen-printed graphene electrode is demonstrated. By using the differential pulse technique, the SSZ produced a well-defined peak of around -0.3 V (versus Ag/AgCl) in Britton-Robinson (BR) buffer pH 4. Supporting electrolytes, pH, and salts all significantly impact SSZ reduction. Therefore, their impact on the working solutions was assessed. We discovered that using a mixture of BR buffer with pH 4 and MgCl2 as a supporting electrolyte can enhance SSZ sensitivity by approximately 1.7 times while simultaneously increasing detection selectivity. Under optimal conditions, the proposed assay demonstrated the ultrasensitive determination of SSZ with a broad linear detection range from 0.01 to 100 µM and a low detection limit of 4.7 nM (S/N = 3). To demonstrate the impact of the proposed method, the sensor has been successfully applied for the quantitative determination of SSZ in pharmaceutical, urine, and artificial serum sample. Therefore, this approach could offer simplicity, and rapidity, and serve as an alternative to the SSZ detection in practical applications.