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The secondary inner filter effect on the fluorescence excitation spectra of rhodamine 6G aqueous solutions with concentration was demonstrated in this paper. The peak of fluorescence excitation spectrum stays at 525 nm at low concentrations, while it breaks up and turns into valley at high concentrations. The threshold concentration was determined to be 3.16 mg/L by the second derivative spectroscopy. A mathematical model was proposed to explain the inflection point of fluorescence excitation spectrum caused by the secondary inner filter effect. On the basis of it, the threshold concentration was calculated to be 2.86 mg/L, approaching to the experimental result. [ABSTRACT FROM AUTHOR]