Due to the epidemics of emerging microbial diseases worldwide, the accurate and rapid quantification of pathogenic bacteria is extremely critical. In this work, a highly sensitive DNA-based electrochemical biosensor has been developed to detect Vibrio cholerae using gold nanocube and 3-aminopropyltriethoxysilane (APTES) modified glassy carbon electrode (GCE) with DNA carrier matrix. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) experiments were performed to interrogate the proposed sensor at each stage of preparation. The biosensor has demonstrated high sensitivity with a wide linear response range to target DNA from 10−8 to 10−14 (R2 = 0.992) and 10−14 to 10−27 molL−1 (R2 = 0.993) with a limit of detection (LOD) value of 7.41 × 10−30 molL−1 (S/N = 5). The biosensor also exhibits a selective detection behavior in bacterial cultures that belong to the same and distant genera. Moreover, the proposed sensor can be used for six consecutive DNA assays with a repeatability relative standard deviations (RSD) value of 5% (n = 5). Besides, the DNA biosensor shows excellent recovery for detecting V. cholerae in poultry feces, indicating that the designed biosensor could become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety, and environmental monitoring.