A sensitive, selective and rapid spectrofluorimetric method is proposed for the determination of thiamine by using mimetic enzyme iron(III) tetrasulfonatophthalocyanine (FeTSPc) as a catalyst for the oxidation reaction between thiamine and hydrogen peroxide. It is based on the oxidation of thiamine in alkaline medium to give an intensively fluorescent compound, which has an excitation wavelength of 375 nm and an emission wavelength of 440 nm. The determination was found to be activated by fluorogenic substrates with a p-hydroxyphenyl structure such as L-tyrosine, tyramine and p-hydroxyphenylpropionic acid. Under optimum conditions, the responses for thiamine were linear from 1.0 × 10^–8 to 1.0 × 10^–4 mol L^–1, with a detection limit of 4.3 × 10^–9 mol L^–1. The relative standard deviation was 2.2% for 2.0 × 10^–7 mol L^–1 thiamine (n = 6). The activation of the p-hydroxyphenyl substrates, the effects of some experimental conditions and the influence of foreign substances were investigated. The potential application of the method was tested by selectively determining thiamine in commercial vitamin B₁, vitamin B complex and rice.