<p>R apid and accurate detection of pathogenic bacteria is crucial for disease diagnosis in clinical practice. The infection of <i>Escherichia coli</i> (<i>E. coli</i>) and <i>Staphylococcus aureus</i> (<i>S. aureus</i>) is closely related to the development and complications of patients with bladder cancer. In this study, an aptamer biochip for <i>E. coli</i> and <i>S. aureus</i> simultaneous detection was developed based on copper single atom material (Cu-SAC) and gold nanoparticles (AuNPs) via high-resolution electrohydrodynamic (EHD) printing technology. The main strategy was based on the specific recognition of aptamer and the signal amplification of AuNPs/Cu-SAC. Following aptamer immobilization, the electrochemical signal gradually decreased. Using this proposed electrochemical aptamer biochip, <i>E. coli</i> and <i>S. aureus</i> can be quantified at a linearity range of 10–1.0 × 10⁸&#xa0;CFU&#xa0;mL⁻¹. The limit of detection (LOD) was as low as 3&#xa0;CFU&#xa0;mL⁻¹. The outstanding signal amplification performance is due to the combination of Cu-SAC and AuNPs, which efficiently supply electrons to Fe³⁺, thereby facilitating the Fe³⁺/Fe²⁺ cycle. Furthermore, employing this portable biochip enables simultaneous measurements of <i>E. coli</i> and <i>S. aureus</i> concentrations in urine and milk samples with acceptable recoveries. This portable biochip holds significant potential for pathogenic bacteria detection&#xa0; in field-based applications. Moreover, it is expected to provide a powerful auxiliary means for the monitoring&#xa0; of&#xa0;bladder cancer patients.</p> Graphical Abstract <p></p>