Rapid and early identification of pathogenic bacteria in food and clinical samples is critical for infectious disease control and precision medicine. In this study, we present a novel Magnetic metal–organic framework (MOF)@Aptamer-based Entropy-driven fluorescence biosensor combined with mesophilic Clostridium butyricumArgonaute (CbAgo) for multiplexed and DNA extraction- and amplification-free detection of pathogenic bacteria, termed the MAEA biosensor. In this study, magnetic zirconium-based MOF nanoparticles were modified with a phosphorylated aptamer as an efficient signal converter, which recognized the pathogens and released the complementary DNA to activate the entropy-driven circuit reaction. The highly efficient catalytic DNA circuit and isothermal enzyme-free format produce large amounts of guide DNA. CbAgo exerts precise and highly efficient endonuclease activity under the guidance of guide DNA, which makes the MAEA biosensor useful for multiplexed and DNA extraction- and amplification-free detection of pathogenic bacteria with high sensitivity (<102 CFU/mL). This study demonstrates a highly selective and reliable magnetic Fe3O4@MOF-@aptamer-based biosensor using CbAgo for simultaneously detecting multiple pathogenic bacteria, providing a potential tool for other infectious disease pathogens with convenient operation and high sensitivity.