A spectrofluorimetric method was established for the quantitative determination of 3-glycidoxypropyltrimethoxysilane (GPTMS), based on the quantitative reaction between dihydroxyl of hydrated epoxy groups and potassium periodate generating formaldehyde and the reaction of the formaldehyde with acetylacetone in the presence of ammonium ions generating a new compound having yellow green fluorescence. The established method was then used to determine the purity of a commercially obtained GPTMS sample and the amount of epoxy groups on GPTMS doped nanosilica composite (GPTMS/nano-SiO2). The conditions such as the concentrations of ammonium salts and acetylacetone, pH value, coloring temperature, and coloring time were investigated in detail with respect to their effects on the fluorescence intensity. It was found that the established method is simple in terms of operation and can be used to practically determine the purity of GPTMS and the amount of epoxy groups on GPTMS/nano-SiO2 with good accuracy and reproducibility.