Herein, we report a multifunctional DNA hydrogel (TBDH) platform that integrates the specific detection and efficient inactivation of S. aureus within a single programmable architecture. The platform consists of three core components: an aptamer (Apt) for target recognition, two DNA signaling strands (SA and SB) for fluorescence transduction, and self-assembled antibacterial nanoparticles (TBNPs). Through rational structural integration, these elements are incorporated into a tunable polymeric network, enabling a unified “recognition–reporting–inactivation” function. In the presence of S. aureus, Apt binding triggers hydrogel dissociation, restoring the quenched Cy3 fluorescence and generating a dose-dependent signal with a detection limit of 11 CFU mL−1. Simultaneously, hydrogel collapse induces on-demand release of TBNPs, which exhibit potent antibacterial and anti-biofilm efficacy (>99.9% inactivation) by disrupting bacterial membrane integrity. The TBDH platform demonstrates high specificity, rapid response (90 min), and reliable performance in complex matrices. This work provides an integrated, stimuli-responsive strategy for simultaneous pathogen detection and eradication, offering promising potential for applications in food safety and clinical infection control.