Antibiotics are crucial due to their widespread use in human and veterinary medicine. Cefotaxime, a third-generation cephalosporin, prevents bacterial production and treats central nervous system infections like meningitis and septicemia. This study developed a novel electrochemical sensor for detecting Cefotaxime (CFTX) by electrodepositing gold-bismuth nanoparticles (Au-Bi NPs) on a carbon paste electrode (CPE). The electrochemical parameters were optimized for the successful deposition of Au-BiNPs. Morphological and X-ray diffraction (XRD) analyses revealed nanoparticle clusters of cubic Au and stacked monoclinic BiNPs on the CPE surface, with crystallite sizes of 7.5 and 6.3 nm, respectively. Cyclic voltammetry (CV) showed that the sensor had superior electrochemical activity for CFTX oxidation compared to bare CPE, BiNPs/CPE, and AuNPs/CPE, mainly due to the synergetic effect of Au-Bi nanoparticles. Electrochemical impedance spectroscopy (EIS) indicated minimal resistance to electron transfer at Au-BiNPs/CPE. Voltammetric investigations revealed a diffusion-controlled electrooxidation process involving two protons and two electrons, influenced by solution pH and scan rate. The sensor displayed excellent analytical parameters for CFTX detection, including a wide linear range (0.5 μM to 20 μM), high sensitivity (4.55 μAμM−1 cm−2), and a low detection limit (0.094 μM). Density Functional Theory (DFT) calculations provided theoretical insights into the sensor’s performance. The sensor also showed excellent reproducibility, repeatability (RSD values of 3.74 % and 1.71 %, respectively), and long-term stability of about one month, offering a new method for accurate and sensitive CFTX detection.