Imaging of bacterial infections can be used for a wide range of investigations, including diagnosis and pathogenesis of infections, and molecular probes targeting biological processes during infection have been used extensively. However, in vivo visualization of bacteria using molecular probes is still challenging due to the difficulty of directly targeting specific bacteria. Here, we propose a new fluorescent nano-bio probe based on a quorum-sensing (QS) antagonist for imaging drug-resistant bacteria. Because most bacteria use QS-based communication to synchronize the regulation of virulence gene expression during infection, QS-based probes can be used to assess the in vivo localization and distribution of pathogenic bacteria. Our developed fluorescent, quorum-based nano-bio probes (QNBPs) target agr-activation in multiple-drug resistant Staphylococcus aureus (MRSA) and were successfully used for in vivo localization of MRSA in sepsis and dermonecrotic animal infection models. Interestingly, we found that QNBPs can diffuse through biofilms and thus provide a new strategy for detecting MRSA embedded within a biofilm. Our findings suggest that our QNBPs have great potential for directly imaging pathogenic bacteria.