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In this work, we report the preparation and characterization of novel dye doped fluorophore Eosin yellowish silica nanoparticles (ESNPs). We synthesized ESNPs by the Stöber method via encapsulation of Eosin Yellowish in silica particles by the condensation of tetraethyl orthosilicate under alkaline condition at room temperature. The resulted ESNPs were characterized by transmission electron microscopy, atomic force microscopy; UV-Visible, fluorescence and Fourier transform infrared spectroscopy. The sizes of the nanoparticles have been found to be 300.0 (±1.0), 400.0 (±1.1) and 500.0 (±5.2) nm depending the reaction conditions under which they were synthesized. Furthermore, because of intense light emission, the ESNPs were used as fluorophore in a peroxyoxalate chemiluminescence system. The effect of solvent and concentrations of necessary reagents, bis(2,4,6-trichlorophenyl)oxalate, sodium salicylate, hydrogen peroxide and the effects of size of the ESNP and temperature on the luminescence efficiency of the system were examined. The activation kinetic parameters of the system were also evaluated from the temperature investigation.