To investigate the application of immunomagnetic dextrin microspheres for rapid isolation and detection of Listeria monocytogenes. The magnetic dextrin microspheres were activated by epichlorohydrin and then crosslinked with antibodies to prepare immunomagnetic dextrin microspheres. The synthesis conditions were optimized by single factor and orthogonal tests. To capture a single extended Listeria monocytogenes using immunomagnetic microspheres synthesized cyclodextrin， under external magnetic field， the magnetic enrichment of immune bacterial DNA extraction， dextrin microspheres， amplified by PCR， agarose gel electrophoresis. The results show that the best condition is the activation reaction of epichlorohydrin dosage is 0.4 mL/g， the concentration of NaOH is 2.0 mol/L， the reaction time is 5 h； the optimal synthesis conditions of preparation of immunomagnetic microspheres is antibody dextrin mass concentration is 1.0 mg/mL， the reaction temperature is 28 ℃， the reaction time is 2.5 h； capture Listeria bacteria the best dosage of immunomagnetic microspheres， dextrin as the best of 100 μL， the capture time is 1 h. The results showed that the immunomagnetic dextrin microspheres could effectively and rapidly capture the single Listeria strain and amplify the specific products. The detection limit could reach 10 single Listeria strains per mL of bacteria.