A novel material for sensing cations in water via fluorescence spectroscopy is presented. The material consists of a glass substrate functionalized with a series of fluorescent self-assembled monolayers. Parallel modification with pairs of fluorophore-binding molecules of monolayers formed on glass yield a library of sensitive glass substrates. Measurements of the changes in fluorescence intensity of the layers upon addition of aqueous solutions of Cu2+, Co2+, Ca2+ and Pb2+ confirmed the ability of the monolayer library to produce a fingerprint response for separate analytes with a high reproducibility. This new protocol for fabrication of sensitive probes in glass is suitable for array fabrication in small size substrates. Additionally, the covalent attachment of the fluorophore moieties to the monolayer allows monitoring of the integrity of the monolayer in time in contact with solutions. To the best of our knowledge this is the first example of sensing of cations in water by a self-assembled monolayer on glass.