An efficient platform for the detection of Salmonella enterica serovar Typhi (S. Typhi) is essential for early-stage diagnosis of typhoid to prevent and contain outbreaks. Here, we fabricated an electrochemical DNA biosensor for selective identification of S. Typhi in real samples. The biosensor has been fabricated by immobilizing an amine labelled S. Typhi specific single-strand capture probe on the surface of gold nanoparticles (AuNP) and poly cysteine (P-Cys) modified screen-printed electrode. Differential pulse voltammetry (DPV) of anthraquinone-2-sulfonic acid monohydrate sodium salt (AQMS) as a signal indicator was monitored to detect S. Typhi by hybridization of target DNA with the probe DNA. The fabricated biosensor shows a detection range of 1 × 10−6 to 1 × 10−22 molL−1 with a LOD of 6.8 × 10−25 molL−1 in S. Typhi complementary linear target and 1.8 × 105 to 1.8 CFUml−1 with a LOD of 1 CFUml−1 in a real S. Typhi sample. The biosensor shows excellent discrimination ability to some bases mismatched and different bacterial cultures (same and distant genera). The most beneficial points of the proposed DNA biosensor are the lower limit of detection and the ability to reuse the biosensor more than 6 to 7 times. In addition, the practicability of the biosensor was investigated via detecting S. Typhi in blood, poultry feces, egg, and milk whereby excellent recoveries ranging from 96.54 to 103.47% were demonstrated indicating that this biosensor might be the most promising diagnostic tool for monitoring S. Typhi in clinical and food samples.