The widespread use of antibiotics has made multidrug-resistant bacteria (MDRB) one of the greatest threats toward global health. Current conventional microbial detection methods are usually time-consuming, labor-intensive, expensive, and unable to detect low concentrations of bacteria, which cause great difficulties in clinical diagnosis and treatment. Herein, we constructed a versatile biosensing platform on the basis of boric acid-functionalized porous framework composites (MOF@COF-BA), which were able to realize highly efficient and sensitive label-free MDRB detection via fluorescence. In this design, MDRB were captured using aptamer-coated nanoparticles and the fluorescent probe MOF@COF-BA was tightly anchored onto the surface of MDRB due to interactions between boric acid groups and glycolipids on bacteria cells. Benefitting from the remarkable fluorescence performance of MOF@COF-BA, rapid and specific detection of MDRB, such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii (AB), was realized with a detection range of 20-108 CFU/mL (for both) and limits of detection of 7 CFU/mL (MRSA) and 5 CFU/mL (AB). The feasibility of using the developed platform to selectively detect MRSA and AB from complex urine, human serum, and cerebrospinal fluid samples was also demonstrated. This work provides a promising strategy for accurate MDRB diagnosis, avoiding serious infection using rational antibiotic therapy.