We have developed a magnetic enzyme-linked immunosorbent assay (MELISA) based poly-L-lysine (PLL) mediated brushes on magnetic nanoparticles (MNPs) for detection of Escherichia coli O157:H7 in river water samples. In the MELISA, we couple PLL on the surface of magnetic nanoparticles to prepare the PLL brushes (MPLLBs). PLL with plentiful amine groups provides multi-binding sites to allow the binding of both horse radish peroxidase (HRP) and antibody (Ab) on the surface of the MPLLBs with high capacity. Compared with the conventional particles (the binding capacity of MNPs for HRP and Ab are 19 mu g/mg and 16 mu g/mg, respectively), MPLLBs have achieved an improvement of 43-fold and 24-fold in binding capacity for HRP (816 mu g/mg, protein mu g per mg of MPLLBs) and Ab (387 mu g/mg), respectively. This multifunctional Ab-MPLLBs-HRP conjugate serves as not only an immune-carrier for magnetic enrichment but also an enzyme assembly for signal amplification system. Compared with the conventional ELISA (the detection of limit is 400 cfu/mL), MELISA shows enhanced sensitivity (8 cfu/mL) and shortened the analysis time (within 2 h) for the detection of Escherichia coli O157:H7 in river water sample, which provides an attractive candidate platform for the rapid and sensitive detection of pathogen in complex samples.