Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive, and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenience. In this study, we developed a G-quadruplex-based CRISPR-Cas12a (clustered regularly interspaced palindromic repeats, CRISPR; CRISPR-associated nuclease, Cas12a) bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella-specific invA gene activated the trans-cleavage activity of Cas12a and triggered CRISPR-Cas12a-based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K+. This DNAzyme could catalyze the TMB-H2O2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas-based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity, and on-site capability.