In this work a new spectrofluorimetric method for the determination of vitamin B(1), based on the catalytic activity of horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2), has been developed. Non-fluorescent vitamin B(1) was easily converted through catalytic oxidation in alkaline medium into a fluorescent compound, even without exposure to light. The linear range for vitamin B(1) observed was 0.026-16.83 microg/mL (RSD = 1.75%). The correlation coefficient for the calibration curve and limit of detection were found to be 0.9964 and 0.015 microg/mL, respectively. The developed method is practical, simple, sensitive and relatively free from interference by coexisting substances and has been successfully applied for the determination of vitamin B(1) in pharmaceutical preparations.