The present work developed an electrochemical genosensor for the detection of virulence outer membrane protein A (ompA,tDNA) gene ofCronobacter sakazakii(C. sakazakii) by exploiting the excellent glucose-oxidase-mimicking activity of copper Metal-organic frameworks (Cu-MOF) doped with gold nanoparticle (AuNPs). The signal nanotags of signal probes (sDNA) that biofunctionalized AuNPs@Cu-MOF (sDNA-AuNPs@Cu-MOF) were designed using an Au-S bond. The biosensor was prepared by immobilization capture probes (cDNA) onto an electrodeposited AuNPs-modified glassy carbon electrode (GCE). AuNPs@Cu-MOF was introduced onto the surface of the GCE via a hybridization reaction between cDNA and tDNA, as well as tDNA and sDNA. Due to the enhanced oxidase-mimicking activity of AuNPs@Cu-MOF to glucose, the biosensor gave a linear range of 1.0 × 10−15to 1.0 × 10−9mol L−1to tDNA with a detection limit (LOD) of 0.42 fmol L−1under optimized conditions using differential pulse voltammetry measurement (DPV). It can be applied in the direct detection ofompAgene segments in total DNA extracts fromC. sakazakiiwith a broad linear range of 5.4−5.4 × 105CFU mL−1and a LOD of 0.35 CFU mL−1. The biosensor showed good selectivity, fabricating reproducibility and storage stability, and can be used for the detection ofompAgene segments in real samples with recovery between 87.5% and 107.3%.