The harm of pathogenic bacteria to humans has promoted extensive research on physiological processes of pathogens, such as the mechanism of bacterial infection, antibiotic mode of action, and bacterial antimicrobial resistance. Most of these processes can be better investigated by timely tracking of fluorophore-derived antibiotics in living cells. In this paper, we will review the recent development of fluorescent antibiotics featuring the conjugation with various fluorophores, and focus on their applica-tions in fluorescent imaging and real-time detection for various physiological processes of bacteria in vivo.