A novel electrochemical glassy carbon electrode modified with a multi-walled carbon nanotube, cytochrome c (Cyt c) and zinc oxide nanoparticles (ZnONPs) was fabricated to increase the sensitivity of electrode for the detection of streptomycin (STN) in certain pharmaceutical samples. Cyclic voltammetry (CV) and differential pulse voltammetry techniques were used for an electrochemical characterization of the electrode. Furthermore, the electrochemical biosensor construction phases were examined by using X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). Under the optimal experimental conditions, the electrode offers a high selectivity and sensitivity signaling in the co-existence method of STN with the linear concentration ranging from 0.02 to 2.2 μM. The detection limits (LOD) and limit of quantification (LOQ) were found to be 0.0028 and 0.0562 μM, respectively. The fabricated sensing electrode has good stability, reproducibility and sensitivity towards STN in the pharmaceutical samples. Preliminary determinations of binding sites within the specified grid box size, which covers both Cyt c and STN, were done by molecular docking analysis. Moreover, density functional theory (DFT) computations were performed to provide insightful information into the optimized geometry of STN.