The emergence of bacteria genetically resistant to first- and second-generation fluoroquinolones has resulted in increased consumption of levofloxacin (LEV) in human and veterinary medicine. In this regard, the development of low cost and good sensitivity electrochemical devices has been highly required. Thus, in this work, we propose the development of a disposable electrochemical device (DED) using a lab-made conductive ink based on graphite powder and nail polish immobilized on a rigid polyvinyl chloride support (transparent sheet). Additionally, a simple and quick protocol for the electrodeposition of silver nanoparticles was used in order to improve the electroanalytical performance of the sensor (2.75-fold). A differential pulse voltammetry (DPV) method was optimized and the sensor was applied for LEV monitoring in pharmaceutical formulation samples, synthetic urine and simulated body fluid. The method showed a wide linear working range ranging from 0.5 to 50 μmol L-1 and a detection limit of 68.3 nmol L-1. Furthermore, the precision was adequate (RSD < 4.7%), while the accuracy was evaluated through spiked samples with percent recovery ranging from 93 to 103%. The sensor was also shown to be selective for LEV against other electroactive antibiotic species, thus demonstrating suitable characteristics for electroanalytical applications.