Voltammetric detection of active substances has occupied an important place in the last decades. In this study, a novel highly efficient electrochemical sensor was fabricated using a combination of titanium dioxide nanoparticles and multi-walled carbon nanotubes mixed with graphene oxide sheets for the sensitive detection of the antibiotic Azithromycin.  The results show that the constructed electrode has excellent electrocatalytic activity for Azithromycin detection (pH 7) compared to the unmodified electrode due to the mobilized TiO₂ nano-conductors on the MWCNTs@GO.  The electrochemical behavior of Azithromycin was perfectly reversible. Transmission electron microscopy, X-ray diffraction, infrared spectroscopy, and Raman spectroscopy analyses were performed to examine the particularities of the IL-TiO₂ NPs@MWCNTs/GO/GCE interface. The effects of pH, accumulation time, scan rate, and the amount of multi-walled carbon nanotubes required for creation were investigated and optimized by applying Cyclic Voltammetry and DPV at pH 7.0. Phosphate buffer medium. The results showed that the number of protons and electrons involved in the electrooxidation reaction of Azithromycin is equal. The calibration curve was plotted in the concentration range of 10^-3 to 0.5×10^-6 M using the DPV method. The limit of detection and limit of quantification were calculated as 1.772×10^-8 M and 5.83×10^-8 M, respectively. The described method was applied to determine Azithromycin in pharmaceutical formulations and human blood and urine samples. The good recovery values between 96.6% and 99.1% suggest the applicability, efficiency, and reliability of the sensor for the determination of Azithromycin.