The pervasive presence of antibiotics in food has severe health impacts due to the rise in antibiotic-resistant bacteria. Therefore, the fabrication of a reliable, sensitive, rapid, and selective platform for antibiotics detection is the required for healthcare services. Here, we aim to fabricate an electrochemical immunosensor based on lanthanum oxide nanoparticles (nLa2O3 NPs) for sensitive and selective determination of trace amounts of ciprofloxacin (CPX) antibiotic. Briefly, the synthesis of nLa2O3NPs was carried out via wet-chemical co-precipitation process and characterized with various techniques using X-ray diffraction, fourier transform infrared spectroscopy, Raman spectroscopy, contact angle, atomic force microscopy, transmission electron microscopy, and electrochemical methods. These nLa2O3 NPs were used for enhancing the electrochemical response of the immunosensor. Further, nLa2O3 NPs were functionalized by APTES and were deposited electrophoretically on the surface of ITO glass substrate, followed by immobilizing anti-CPX antibodies covalently via EDC-NHS chemistry. Blocking of the non-specific area was done with the help of BSA. The change in electrochemical response studies of the developed immunosensor (BSA/anti-CPX/APTES/nLa2O3/ITO) was monitored to detect CPX. Under optimized conditions, studies showed that the developed biosensor had a wide linear detection range of 0.001–0.5 ng/mL and 1–1000 ng/mL, with the sensitivities of 11.44 μA ng-1 mL cm-2 (R2 = 0.968), and 7.88 μA ng-1 mL cm-2 (R2 = 0.972), respectively and having lower detection limit of 0.001 ng/mL. The developed immunosensor showed good reproducibility, repeatability, sensitivity, and stability that was effectively explored to detect CPX in milk samples.