The rise of resistance to all known antibiotics is a global crisis. In addition to novel treatment options, there is an urgent need to develop rapid, specific, sensitive, and reliable diagnostic methods to detect pathogenic bacteria in clinical samples and reduce the overuse and misuse antibiotics. Pseudopaline, a metallophore produced by the human pathogen Pseudomonas aeruginosa, transports divalent metal ions via a dedicated active transport system, making it an ideal carrier for a second functional moiety. In this work, we have developed a pseudopaline fluorescein-conjugated probe (P-FL), able to specifically detect P. aeruginosa in samples (in vitro) among several bacterial species, mammalian cells, or in mouse stomach tissue sections. By replacing the fluorescein with the near-infrared fluorophore, Cyanine-7 (Cy-7) to obtain a pseudopaline-Cyanine-7 (P-Cy7), we showed that P. aeruginosa infections could also be detected specifically in a mouse model (in vivo) using this probe. The remarkable selectivity of these pseudopaline fluorescent probes is due to pseudopaline-mediated metal transport system, exclusively specific to P. aeruginosa. Therefore, our results show that pseudopaline-based probes might provide a new approach to develop fast and effective diagnostics of P. aeruginosa infections.