A method for coupling single-chain antibody fragments (scFvs) to immunomagnetic beads (IMBs) was developed and evaluated using scFvs specific for Listeria monocytogenes. A plasmid vector, pBAD380, was constructed that allowed the expression of histidine-tagged biotinylated scFvs in Escherichia coli. The gene encoding a scFv specific for L. monocytogenes was cloned into pBAD380 and the 6-histidine-tagged biotinylated anti-L. monocytogenes scFvs were coupled to streptavidin-coated IMBs. The ability of the anti-L. monocytogenes scFv-IMBs to capture L. monocytogenes and other Listeria species was evaluated in comparison to commercially available anti-Listeria IMBs. The anti-L. monocytogenes scFv-IMBs displayed higher efficiencies of capture (1.38-19.04%) for most strains of L. monocytogenes than were observed for the anti-Listeria IMBs (0.05-3.35%); also, the anti-L. monocytogenes scFv-IMBs exhibited improved specificity for L. monocytogenes as determined by cell capture efficiency in pure and mixed cultures.