A very sensitive, selective, and rapid spectrophotometric method for the determination of vitamin B1 in the presence of vitamins B2, B6, and B12 was carried out in the UV region without performing a previous separation. Vitamin B1 was selectively and strongly sorbed on Sephadex CMC-25 ion exchanger at pH 4.80 and its intrinsic absorbance measured directly in the solid phase at 247 and 320 nm. The apparent molar absorptivity ranged from 5.63 × 105 to 2.19 × 107 L mol-1 cm-1, depending on the sample volume used (from 10 to 1000 mL). A procedure (for five different sample volumes) was here proposed. Thiamin could be determined in the concentration ranges 0.5−5.5 μg mL-1 (10-mL sample volume) up to 12 −170 ng mL-1 (1000-mL sample volume) with detection limits ranging from 80.0 to 3.0 μg L-1 and RSDs from 1.5 to 2.6%, respectively. The method showed an excellent selectivity against other compounds of the B complex with similar spectral features and which did not cause interference. The method was satisfactorily applied to the determination of vitamin B1 in pharmaceutical preparations.