<p><i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>) is a significant opportunistic pathogen associated with nosocomial infections, particularly in pediatric populations, where it can lead to severe clinical manifestations, including <i>P. aeruginosa</i>—associated meningitis. To meet the critical need for highly sensitive detection of <i>P. aeruginosa</i>, a novel fluorescent biosensor utilizing a three-way junction (TWJ) probe has been developed. This biosensor capitalizes on the specific binding interaction between P. aeruginosa and a tailored aptamer embedded within a DNA TWJ structure. Upon target binding, the double-stranded DNA branches of the TWJ undergo a conformational rearrangement, resulting in the formation of two distinct DNA “Y” junction structures. These structures are subsequently linked by a designed sequence, initiating a DNA polymerase/endonuclease-mediated strand displacement amplification process. The TWJ-based biosensor offers several key advantages: (i) the integration of the aptamer sequence within the TWJ probe ensures high specificity for target recognition, and (ii) the subsequent enzymatic amplification significantly enhances the sensitivity of detection. Under optimized experimental conditions, the biosensor demonstrated a broad linear detection range from 10 to 10 to 10⁵&#xa0;cfu/mL, with an exceptionally low limit of detection of 4.12&#xa0;cfu/mL. Recovery studies further confirmed the reliability and robustness, highlighting its potential for clinical implementation. This innovative bio-sensing strategy represents a significant advancement in diagnostic technology, offering a promising tool for the early and accurate detection of infectious diseases in pediatric patients, with potential applications in improving clinical outcomes.</p> Graphical abstract <p></p>