In order to explore novel NIR fluorescent probes for optical imaging in biomedicines, one desferrioxamine (DFO)-bearing NIR fluorescent probe was designed and synthesized based on a dicarboxylic acid-containing carbocyanine (Cypate). Similar to the free DFO, the resulting conjugate Cypate-DFO showed high binding affinity with Fe(III) and Ga(III) as identified by ES-MS. Nevertheless, the iron binding was found to quench its fluorescent emission significantly, suggesting that the siderophore moiety might perturb the spectroscopic properties of the attached carbocyanine fluorophore through metal binding. As observed by fluorescence microscopy, Cypate-DFO showed significant cellular internalization in A549 cells in vitro. Further studies on novel Cypate-DFO derivatives of this type may reveal some exciting properties and biological activities.