The aim of this study was to establish an IMS-MS/SPR technique for the detection of Staphylococcus aureus (S. aureus) and Staphylococcus protein A (SPA) at the same time, which consists of isolating S. aureus and trapping-enrichmenting its SPA by IMS, and the end point is determined by using either MS or SPR measurements. Magnetic bead (MB) containing aldehyde group was synthesized with latex-polymerization and immunomagnetic bead (IMB) was fabricated by modifying its surface with an oriented layer of human IgG in covalent linkage. As soon as sample of pulverator-treated bacterial cell lysate (108 cfu/mL) was incubated with IMB at 4 ◦C for 30 min, SPA was captured and separated from the mixed solution in a few minutes by the IMB and then detected with mass spectrometry after washing. SPR was used to detect S. aureus quantitatively in situ at the end-detection procedure. All in all, this technique can be employed to detect rapidly SPA and S. aureus within 2 h and also be applied to detect other cells or their membrane proteins with changed modified antibodies.