A novel electrochemical biosensor for detecting pathogenic bacteria was designed based on specific magnetic separation and highly sensitive click chemistry. Instead of enzyme-antibody conjugates, organic-inorganic hybrid nanoflowers [concanavalin A (Con A)-Cu3(PO4)2] were used as the signal probe of the sandwich structure. The inorganic component, the copper ions of hybrid nanoflowers, was first used to amplify signal transduction for enzyme-free detection. Sodium ascorbate could dissolve Cu3(PO4)2 of the signal probe to produce Cu2+, which was subsequently converted to Cu+, triggering the Cu+-catalyzed alkyne-azide cycloaddition (CuAAC) reaction between azide-functionalized ssDNA (a fragment of the DNAzyme-containing sequence) and alkyne-functionalized ssDNA immobilized onto the electrode surface. As a result, the DNAzyme was immobilized onto the gold electrode, which produced a positive and stable electrical signal. An exceptional linear relationship was observed between the electrical signal and the concentration of Salmonella typhimurium (101-107 CFU mL-1) with a detection limit of 10 CFU mL-1. The developed electrochemical biosensor based on dual signal amplification of Cu3(PO4)2-mediated click chemistry and DNAzymes exhibited good results in detecting S. typhimurium in milk samples.