In the present study, a synchronous spectronfluorimetric method has been applied to the simultaneous determination of vitamin B1, B2 and B6. However, their spectra are overlapped so it is difficult to determine them individually. The parallel factor analysis (PARAFAC) was applied to the resolution of the overlapped spectra of mixtures of vitamin B1, B2 and B6. The delta wavelength range used was from 20 to 120 nm, and the excitation wavelength range was selected from 200 to 500 nm. From the measured fluorescence data a four-factor PARAFAC model was established, and was applied to the determination of a set of synthetic validation samples and some commercial samples with satisfactory results. This work shows that PARAFAC method is a powerful chemometric tool to the determination of mixtures of vitamin Bl, B2 and B6.