Introduction: Tuberculosis (TB) is a significant cause of morbidity and mortality worldwide. The patient compliance with the long treatment regimens is essential for successful eradication. Pyrazinamide (PZA) shortens these regimens from 9 to 6 months, and therefore, improves treatment completion rates. Although PZA is a first-line medication for the treatment of TB, no simple or reliable assay to determine PZA resistance is yet available. In the presence of PZA, only susceptible Mycobacterium tuberculosis strains release pyrazinoic acid (POA). Therefore, the measurement and quantification of released POA is an indicator of PZA resistance. Methods: Two electrochemical sensors were constructed and tested with alternative working electrodes in conjunction with a portable potentiostat to measure the current produced when a potential difference of 2 V is applied to varying concentrations of POA in controlled solutions. Results: The large (13.2 mm) electrochemical sensor was able to detect POA at a minimum concentration of 40 μM to a statistically significant level (P = 0.0190). Similar graphical trends were obtained when testing the electrochemical sensor in the supernatant of a negative microscopic observation drug susceptibility assay culture, irrespective of the presence of PZA. Conclusion: Inexpensive and reusable electrochemical sensors with a portable potentiostat are a promising tool for the detection of POA, a biomarker of PZA susceptible M. Tuberculosis.