We evaluated the cell wall binding (CWB) domain of Staphylococcus aureus autolysin as an affinity reagent for bacteria. A fusion of CWB domain and green fluorescent protein (CWB-GFP) bound to S. aureus with a dissociation constant of 15 nM. CWB-GFP bound to a wide range of gram-positive bacteria, but not to most gram-negative bacteria. We suspected that the outer membrane of gram-negative bacteria inhibits the access of CWB-GFP to peptidoglycan layer. Indeed, CWB-GFP bound to gram-negative bacteria when they were treated with benzalkonium chloride. Because CWB-GFP bound to the bacterial peptidoglycan layer, it appeared to be an effective affinity reagent for bacteria and CWB fusion with reporter proteins could be applied to detect bacteria. We also constructed a fusion of CWB and luciferase, which can be used for the rapid detection of bacteria.