The development of a flow-through solid-phase room temperature phosphorescence (RTP) method for the sensitive determination of orthophosphate in aqueous samples, based on the energy transfer from a phosphor molecule (acting as a donor) to an orthophosphate dye-indicator (acting as an acceptor) is described. The proposed method, to our knowledge the first RTP optosensor for orthophosphate developed so far, is based on the injection in a flow system of 1ml sample treated to form phosphomolybdenum blue from the orthophosphate. After injection, the phosphomolybdenum blue is on-line co-immobilised onto a polymeric resin containing adsorbed erythrosine B. This selected donor molecule exhibits strong RTP in a de-oxygenated aqueous media when retained on the surface of polymeric resin beads. Absorption spectra of the phosphomolybdenum blue possess a desirable spectral overlap with the emission spectra of the RTP donor and a non-radiative energy transfer occurs from the phosphor molecule to the acceptor dye. An increase in the concentration of orthophosphate of the solution causes an absorption increase of the acceptor (phosphomolybdenum blue) and, therefore, an increase in the energy transfer, which brings about a decrease of the RTP emission. After measurement, the active sensing phase can be regenerated by passing 2ml of 2M sodium hydroxide plus 2ml of methanol. After the injection of 1ml of 2x10(-6)M erythrosine B the system is prepared again for a new sample injection. Potential interferences by ions present in natural waters, which could affect the optosensor response, and analytical performance characteristics of the RTP method are discussed in detail. An orthophosphate detection limit of 0.5ngml(-1) (for 1ml sample injection volume) was achieved. Finally, the selected RTP flow-through optical sensor has been successfully tested for the determination of orthophosphate in different water samples at a very few ngml(-1) levels.