Layered transition metal dichalcogenides (TMDs), with an extensive surface area, intriguing tunable electrical and optical features, and a distinctive Van der Waals layered structure, yield outstanding sensing properties. Essentially, most TMDs originally existed in the crystallographic phase of a 2H trigonal prismatic structure, which is semiconducting in nature with poor electrocatalytic activity. In contrast, vanadium diselenide (VSe2) with its metastable metallic 1 T octahedral crystal structure has been proven to be an outstanding electrode material, embracing exceptional electrocatalytic behavior for various electrochemical (EC) applications. However, practically, VSe2 has hardly ever been explored in the field of biosensing technology. This study presents a novel EC biosensor based on the antibody of Salmonella Typhimurium (Anti‐ST) immobilized on VSe2‐supported Indium tin oxide (Anti‐ST/VSe2/ITO) for quantitative and efficient Salmonella Typhimurium (ST) detection. The Anti‐ST/VSe2/ITO bioelectrode displayed a linear relationship with ST concentration (1.3 × 10–107 CFU/ml) with a limit of detection (LOD) (0.096 CFU/ml) that is lower than previously reported ST biosensors and impressively high sensitivity (0.001996 μA.mL/CFU). Furthermore, the proposed electrode's electroanalytical activity was evaluated in spiked sugarcane juice, demonstrating distinguished applicability for specific ST detection in real samples.