<p>Rapid and reliable detection of multidrug-resistant bacteria is essential for effective clinical management. In this study, we have developed a dual-mode aptasensing platform based on a core–shell MOF-on-MOF structure (UiO-66-NH₂@MOF-919) for the subtype-specific detection of carbapenem-resistant <i>Klebsiella pneumoniae</i> strains carrying NDM-1 and KPC-2. This bifunctional system integrates the peroxidase-like activity of the MOF-919 shell with the fluorescence quenching capability of the UiO-66-NH₂ core, enabling both colorimetric and fluorometric detection within a single platform. Benefiting from this design, the method achieves sensitive and selective detection over a wide linear range from 10 to 10⁸ CFU mL⁻¹, with low detection limits of 7 CFU mL⁻¹ for NDM-1 KP and 4 CFU mL⁻¹ for KPC-2 KP. The proposed platform exhibits good selectivity against non-target bacteria and strong anti-interference capability. Spiked recovery experiments in human serum, urine, and bronchoalveolar lavage fluid yielded recoveries ranging from 93.0% to 110.0% with RSDs below 5.0%, demonstrating good analytical accuracy and applicability in complex biological samples. Compared to conventional aptasensing systems, this approach enables dual-subtype detection within a single platform, improving analytical efficiency and practicality. This work provides a feasible strategy for MOF-on-MOF-based biosensing systems and shows promising potential for rapid clinical detection of drug-resistant bacteria.</p> Graphical abstract <p></p>