Fluorometric determination of thiamine requires the conversion of the analyte to fluorescent thiochrome by hexacyanoferrate(III) oxidation in alkaline solution and the isolation of the produced thiochrome from the reaction medium by solvent extraction. It was observed that thiochrome could be concentrated and separated from the reaction medium by solid-phase extraction. The thiochrome sorpted on the surface of octadecyl-alkylated poly-[styrene/divinylbenzene] (C18-PS/DP) microbeads emitted strong fluorescence upon excitation, the maximum excitation and emission wavelengths being 385 nm and 433 nm, respectively. Based on this observation, a sequential injection renewable surface solid-phase spectrofluorometry was developed for the determination of thiamine. A sequential injection system on-line coupled to a chip-based flow-through cell was employed to handle the chemical reaction, bead injection and discharging, and adsorption of thiochrome. Solid-phase fluorometric detection was realized by coupling the chip-based flow-through cell to a spectrofluorometer with a multistrand bifurcated optical fiber. Under the optimized condition, a detection limit of 0.03 μg ml^-1 was achieved at the sample throughput of 30 h^-1 and consumption of 1 mg C18-PS/DP microbeads for each run. Eleven runs of a 2 μg ml^-1 thiamine standard solution gave a relative standard deviation of 1.0%. The developed approach was successfully applied for the determination of thiamine contents in pharmaceutical preparations.