<p><i>Acinetobacter</i> <i>baumannii</i> is an opportunistic nosocomial bacterial pathogen that causes significant global morbidity and mortality. A rapid and accurate detection platform is essential to minimize the risk of nosocomial infections and drug resistance. We developed an innovative electrochemical biosensor for the rapid detection of <i>A. baumannii</i> in real samples, using a unique enzyme immobilization method. The biosensor is capable of identifying <i>A. baumannii</i> through the specific phages attached to HRP@Cu₃(PO₄)₂ hybrid nanoflower with the help of AuNPs, creating a bifunctional complex that offers both biomolecular recognition and signal amplification. The developed phage-based electrochemical biosensor was efficient in detecting live <i>A. baumannii</i> across a range of 5.77 × 10²–5.77 × 10⁷&#xa0;CFU/mL, up to 3&#xa0;CFU/mL, within 30&#xa0;min in serum and plasma samples. The biosensor employs a straightforward design, offering more simple operation and higher specificity than traditional methods, such as culturing. Furthermore, the immobilization of HRP on the hybrid nanoflowers improved its stability against environmental conditions and affinity for the substrate. The findings of this study demonstrate that electrochemical biosensors based on HRP-hybrid nanoflowers and phages can be effectively utilized for the rapid detection of <i>A. baumannii</i> while also laying the groundwork for stable HRP immobilization.</p> Graphical Abstract <p></p>