The diagnosis of bacterial infections remains a major challenge in medicine. Optical imaging of bacterial infection in living animals is usually conducted with genetic reporters such as lightemitting enzymes or fluorescent proteins. However, there are many circumstances where genetic reporters are not applicable, and there is an urgent need for exogenous synthetic probes that can selectively target bacteria. Optical imaging of bacteria in vivo is much less developed than methods such as radioimaging and MRI. Furthermore near-infrared (NIR) dyes with emission wavelengths in the region of 650·900 nm can propagate through two or more centimeters of tissue and may enable deeper tissue imaging if sensitive detection techniques are employed. Here we constructed an antimicrobial peptide fragment UBI29-41-based near-infrared fluorescent imaging probe. The probe is composed of UBI29-41 conjugated to a near infrared dye ICG-Der- 02. UBI29-41 is a cationic antimicrobial peptide that targets the anionic surfaces of bacterial cells. The probe allows detection of Staphylococcus aureus infection (5×10⁷cells) in a mouse local infection model using whole animal near-infrared fluorescence imaging. Furthermore, we demonstrate that the UBI29-41-based imaging probe can selectively accumulate within bacteria. The significantly higher accumulation in bacterial infection suggests that UBI29-41-based imaging probe may be a promising imaging agent to detect bacterial infections.