Levofloxacin (LF) is a medically important antibiotic drug that is used to treat a variety of bacterial infections. In this study, three highly sensitive and selective carbon paste electrodes (CPEs) were fabricated for potentiometric determination of the LF drug: (i) CPEs filled with carbon paste (referred to as CPE); (ii) CPE coated (drop-casted) with ion-selective PVC membrane (referred to as C-CPE); (iii) CPE filled with carbon paste modified with a plasticizer (PVC/cyclohexanone) (referenced as P-CPE). The CPE was formulated from graphite (Gr, 44.0%) and reduced graphene oxide (rGO, 3.0%) as the carbon source, tricresyl phosphate (TCP, 47.0%) as the plasticizer; sodium tetrakis[3,5-bis(trifluoromethyl)phenyl] borate (St-TFPMB, 1.0%) as the ion exchanger; and levofloxacinium-tetraphenylborate (LF-TPB, 5.0%) as the lipophilic ion pair. It showed a sub-Nernstian slope of 49.3 mV decade−1 within the LF concentration range 1.0 × 10−2 M to 1.0 × 10−5 M, with a detection limit of 1.0 × 10−5 M. The PVC coated electrode (C-CPE) showed improved sensitivity (in terms of slope, equal to 50.2 mV decade−1) compared to CPEs. After the incorporation of PVC paste on the modified CPE (P-CPE), the sensitivity increased at 53.5 mV decade−1, indicating such improvement. The selectivity coefficient (log KLF2+,Fe+3pot.) against different interfering species (Na+, K+, NH4+, Ca2+, Al3+, Fe3+, Glycine, Glucose, Maltose, Lactose) were significantly improved by one to three orders of magnitudes in the case of C-CPE and P-CPE, compared to CPEs. The modification with the PVC membrane coating significantly improved the response time and solubility of the LF-TPB within the electrode matrix and increased the lifetime. The constructed sensors were successfully applied for LF determination in pharmaceutical preparation (Levoxin® 500 mg), spiked urine, and serum samples with high accuracy and precision.