Immunomagnetic beads (IMBs) have been widely used to capture and isolate target pathogens from complex food samples. The orientation of the antibody immobilized on the surface of magnetic beads (MBs) is closely related to the effective recognition with an antigen. We put forward an available strategy to orient the antibody on the surface of MBs by changing the charged amino group ratio of the reactive amino groups at optimal pH value. Quantum dots labeling antigen assay, antigen-binding fragment (Fab) accessibility assay and lysine mimicking were used for the first time to skillfully illustrate the antibody orientation mechanism. This revealed that the positively charged ε-NH₂ group of lysine on the Fc relative to the uncharged amino terminus on Fab was preferentially adsorbed on the surface of MBs with a negatively charged group at pH 8.0, resulting in antigen binding sites of antibody fully exposed. This study contributes to the understanding of the antibody orientation on the surface of MBs and the potential application of IMBs in the separation and detection of pathogenic bacteria in food samples.