A rapid and sensitive fluorimetric method has been developed for the determination of trace hydrogen peroxide (0–5.0 × 10−6mol/l) and, indirectly, glucose (3.0 × 10−7– 3.5 × 10−5mol/l), based on the catalytic effect of hemin on the thiamine, a new fluorogenic substrate, and hydrogen peroxide system at pH=8.0. Using the fixed time method, the detection limits of hydrogen peroxide and glucose are 3.0 × 10−8mol/l and 3.0 × 10−7mol/l, respectively. This method is simple, inexpensive and has been applied successfully to the determination of glucose in human sera with relative standard deviation of 2.4–3.1%.