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In this study, a sensitive immunoassay using immunomagnetic nanobeads (MNBs), manganese dioxide nanoflowers (MnO 2 NFs) and quantum dots (QDs) was developed for simultaneous detection of E. coli O157: H7 and Salmonella typhimurium. MnO 2 NFs were synthesized, functionalized and incubated with QDs to obtain QDs@MnO 2 nanocomposites, followed by modification with antibodies (pAbs) to obtain pAb-QDs@MnO 2 nanocomposites (QM NCs). Target bacteria were first conjugated with MNBs and QM NCs to form MNB-bacteria-QM complexes. Then, QDs were quickly released from the complexes using glutathione to reduce MnO 2 to Mn 2+ . Finally, fluorescent intensity at characteristic wavelength was measured by optical detector to determine target bacteria. This immunoassay could simultaneously and quantitatively detect E. coli from 1.5 × 10 1 to 1.5 × 10 6  CFU/mL with detection limit of 15 CFU/mL and Salmonella from 4.0 × 10 1 to 4.0 × 10 6  CFU/mL with detection limit of 40 CFU/mL in 2 h. The mean recovery for both bacteria in spiked chicken samples was ∼96%., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)