Bumetanide (BMT) is a diuretic that masks the presence of other banned substances in urine or enables athletes to shed excess weight rapidly by increasing water and electrolyte excretion; consequently, BMT is included in the World Anti-Doping Agency's (WADA) list of banned substances, and its detection is crucial for safeguarding the integrity of sports. This study describes a new electrochemical sensor (Ni-TiO<inf>2</inf>-PANI/LabSPE) based on a laboratory-made screen-printed electrode (LabSPE) modified with Ni-doped TiO<inf>2</inf> nanofiber (Ni-TiO<inf>2</inf>) and polyaniline (PANI) for sensitive and selective detection of BMT. The contribution of Ni doped on TiO<inf>2</inf> to the sensing performance of Ni-TiO<inf>2</inf>-PANI/LabSPE was examined in detail by CV, DPV, and EIS techniques. After optimizing the Ni-doping ratio, Ni-TiO<inf>2</inf> concentration, PANI concentration, and film thickness, Ni-TiO<inf>2</inf>-PANI/LabSPE exhibited two linear DPV response ranges for BMT spanning 0.1–20 μM and 20–225 μM, with sensitivities of 0.2638 and 0.0971 μA μM⁻¹, respectively, and an exceptionally low detection limit of 0.03 μM, surpassing all previously reported BMT electrochemical sensors. The sensor also displayed high repeatability (RSD 2.52%), reproducibility (RSD 2.89%) and stability (88% signal retention after 30 days), together with good selectivity towards potential interfering compounds/ions. Application of Ni-TiO<inf>2</inf>-PANI/LabSPE to pharmaceutical and synthetic urine samples yielded recoveries of 91.36–103.35%, in close agreement with a reference spectrophotometric method, confirming its reliability for real-sample analysis.