<p><i>Staphylococcus aureus</i> (<i>S. aureus</i>) is a foodborne pathogen threatening public health due to the lack of rapid and sensitive detection methods for timely identification of its contamination. Surface-enhanced Raman scattering (SERS) shows great potential in quick bio-detection, but its pathogen detection analysis is challenged by limited size-matched substrates for both pathogen separation and SERS enhancement. Here, we designed an MXene-incorporated interconnected porous heterostructure (MIIPH) for effective capture and SERS detection of <i>S. aureus</i>. The MIIPH is fabricated through a one-pot hybridization process that incorporates single Ti₃C₂T_x MXene layers during the construction of the interconnected porous hydrogel. It enables <i>S. aureus</i> capture through both Ti-phosphate coordination and the boronic acid-diol binding interactions. Notably, owing to its highly uniform interconnected porous nanoarchitecture, MIIPH enhances the retention of <i>S. aureus</i> via a spatial confinement effect. Combined with a SERS-active silver probe (Ag-Apt), <i>S. aureus</i> is encapsulated within MIIPH and coated with Ag-Apt, enabling stable encapsulation of <i>S. aureus</i> within the interconnected porous network, while also providing synergistic SERS enhancement from both the embedded MXene component in MIIPH and silver plasma. This platform achieves <i>S. aureus</i> capture and determination with good sensitivity (a detection limit of 151 CFU/mL) and high specificity across a wide linear range (1.51 × 10³1.51 × 10⁸ CFU/mL). Furthermore, the method was successfully applied to the analysis of rice samples. Our approach not only provides a determination tool for <i>S. aureus</i> in complex matrices but also offers a versatile guideline for designing pathogen-specific SERS substrates.</p> Graphical Abstract <p></p>