Objective： To preparation a Listeria monocytogenes specified phage-display single-chain antibody fragments immuno-magnetic beads （scFv-IMBs）， the effectiveness of scFv-IMBs was evaluated by artificial contamination. Method： the capture efficiency and the ability of scFv-IMBs for separating L. monocytogenes from L. innocua in frankfurters was compared to commercially available anti-Listeria immunomagnetic beads （Dyna-IMBs） using qualitative and quantitative culture method， as well as PCR method， in various enrichment systems. Result： the capture efficiency of scFv-IMBs for L. monocytogenes was 15%-55%， which is better than the efficiency of Dyna-IMBs （0.85%-1.27%）， and scFv-IMBs shows good specificity. The capture efficiency of those IMBs in MOPS-BLEB could reach 62%-88%， which was better than IMBs in UVM media （＜5%） and FB media （＜53%）. Moreover， the scFv-IMBs displayed higher specificity to L. monocytogenes （P＜0.01） even when the initial cells concentration of L. innocua were 1 000 times more than that of L. monocytogenes spiked in commercial frankfurters exudates. And the detection limitation combined with scFv-IMBs could reach 1 CFU/mL. Conclusion： scFv-IMBs have better specificity and separation ability than Dyna-IMBs， which could significantly lower the interference of L. innocua in complex matrix.