This study aimed to guide future sensor studies against other pharmaceutical drugs by synthesizing various metallic nanoparticles (NPs). Side damage caused by excessive accumulation of tuberculosis drugs in the body can cause clots in the organs, cause serious damage such as heart attack and respiratory failure, and threaten human life. Therefore, the development of sensors sensitive to various antibiotics in this study is important for human health. In this study, the sensitivity of Fe₃O₄ NPs to tuberculosis drug (rifampicin) was evaluated by catalytic reaction using bare/GCE, MWCNT/GCE, Fe₃O₄NPs@MWCNT/GCE electrodes. First of all, Fe₃O₄ NPs were successfully synthesized for the study and MWCNT/GCE and Fe₃O₄ NPs@MWCNT/GCE electrodes were formed with the modification of the MWCNT support material. It was observed that the Fe₃O₄ NPs@MWCNT/GCE electrode gave the highest signal against the other electrodes. The morphological structure of Fe₃O₄ NPs was determined by various characterization techniques such as Transmission Electron Microscopy, Fourier Transmission Infrared Spectroscopy (FTIR), ultraviolet–visible (UV–Vis), and X-ray differential (XRD) and the obtained NPs were used for sensor studies, and it was observed that the current intensity increased as the scanning speed of each electrode increased in CV and DPV measurements. The particle size of in Fe₃O₄ NPs was found to be 7.32 ± 3.2 nm. The current density was observed in Fe₃O₄ NPs/@MWCNT/GCE. Anodic current peaks occurred in the linear range of 2–25 μM. According to the results obtained from the measurements, the limit of detection (LOD) value was calculated as 0.64 μM limit of quantification (LOQ) 1.92 μM.