Highly sensitive and selective nanosensor for labile iron pool (LIP) determination, has been designed and prepared by immobilization of Fluoresceine-Desferrioxamine (Fl-DFO), a bifunctional fluoro-siderophore probe molecule with great affinity for iron ions (pKf=30.7), into highly ordered mesoporous silica structure. Different immobilization methods of Fl-DFO molecules, such as their encapsulation in surfactant micelles used as templating agents for the synthesis of mesoporous silica, direct impregnation into the mesochannels of as-synthesized mesoporous silica and their surface anchoring by covalent binding with propylamine groups implanted by post-synthesis on the internal surface of mesochannels, have been explored. Each nanohybrid has been fully characterized by small angle XRD, TEM, SEM, solid state Si and C MAS NMR and N adsorption-desorption. The fluorescence properties of nanohybrids obtained have been correlated with the immobilization methods, generating interesting information concerning the localization of Fl-DFO molecules in the channels of mesoporous silica. The leaching of Fl-DFO molecules from mesoporous materials has been investigated. The nanosensor prepared by surface anchoring of Fl-DFO at the internal surface of mesochannels showed high performances with no leaching effect and high sensitivity in regards to its responses to ferric ions. Its fluorescence intensity decreased as soon as first Fe ions are in contact with this nanosensor. A linear relationship between the fluorescence intensity and the ferric ions concentration was observed in low micromolar range. The selectivity of this nanosensor towards other metal ions has also been tested and shown its high affinity to ferric ions. This study can allow the design of a stable, portable, simple, regenerable and cost-effective nanosensor highly sensitive and selective for iron ions with detection limits in the range of cellular LIP in cells, e.g. lower micromolar range.