Staphylococcus aureus (S. aureus) is one of the pathogens that seriously endanger human health and are widespread in nature. Food poisoning incidents caused by S. aureus frequently break out. Therefore, it is significant that a rapid and accurate method is established for S. aureus detection. In this study, a strategy using magnetic separation combine with fluorescence analysis was proposed for S. aureus detection. Boric acid can selectively enrich compounds with cis-diol groups through a reversible esterification reaction, and has been widely used in the identification of sugars and glycosylated molecules. As a boric acid derivative, 3-aminophenylboronic acid (APBA) has the advantages of stability, low cost, easy synthesis, etc. Therefore, using APBA as a recognition molecule to prepare magnetic nanomaterials can achieve the enrichment of S. aureus. In order to reduce steric hindrance and ensure the dominant orientation of APBA, thus improving the enrichment performance of target bacteria. The polyethylene glycol (PEG) was used as a mediator to prepare PEG-mediated APBA functionalized magnetic nanomaterials (APBA-PEG-MNs). In addition, this study used FITC-pig IgG to label S. aureus because the Fc fragment of IgG to bind protein A on the surface of S. aureus and realized the detection of S. aureus by detecting fluorescence. Under the optimal experimental conditions, the capture efficiency of APBA-PEG-MNs for S. aureus with the concentration of 2.7 x 10(6) CFU/mL in spinach, juice and pool water spiked samples was 92.6%, 91.6%, 91.6%. The limit of detection of the strategy in spiked samples was 2.7 x 10(2) CFU/mL within 80 min.