Transparent mesoporous silica microspheres were successfully synthesized via sol-gel method followed by calcination. The prepared microspheres consisted of sub-micrometer particles embedded in a gel matrix, which was the main product of the fast hydrolysis and condensation of TEOS in the octylamine-containing medium. The continuous gel network gave light transparency of these microspheres. The microspheres were statically immersed in rhodamine 6 G (R6G) dye solutions with different concentrations. The guest R6G molecules loaded in the microspheres exhibited significantly enhanced fluorescence performance compared with the correspondent dye solutions. The localization and dispersion of dye molecules in the nanopores of microspheres reduced the aggregation of dye molecules which otherwise quenched the luminescence. A comparative study on the loading of the same dye within another transparent and an opaque silica gel with larger pore sizes indicated that the transparency and the smaller pore size of the transparent microspheres explained its larger fluorescence enhancement effect.