In this study, vancomycin (Van) functionalized PEGylated-magnetic nanoparticles(Van-PMs) combined with polymerase chain reaction (PCR) showed great potential for highly efficient detection of L. monocytogenes at low infectious dose. Herein, Van-PMs played as universal molecular probes for targeting bacterial cells, while PEG was used to help Van close to D-alanyl-d-alanine (D-Ala-d-Ala) moieties on surface of L. monocytogenes. Under optimum conditions, the developed Van-PMs complexes exhibited extremely high capture efficiency (CE) in a 10 mL bacterium suspension with the value was higher than 90% and 83% in the phosphate-buffered saline (PBS) solution and lettuce samples, respectively. Meanwhile, Gram-negative bacteria slightly influenced CE even under concentrations as high as 106 CFU/mL. The assay of Van-PMs-PCR was applied to detect L. monocytogenes with detection limits of 30 CFU/mL and 30 CFU/g in PBS solution and lettuce samples, respectively. The whole experiment was completed within 4 h. This assay demonstrated the potential of this modified method in practical application involving the detection of L. monocytogenes or other Gram-positive pathogenic bacteria.