The determination method of L-phenylalanine (LPA) by fluorescence quenching was developed. The assay was based on the combination of the cucurbit[7]uril (CB[7]) with palmatine hydrochloride (PAL) reaction. In the presence of CB[7], the fluorescence of PAL was quenched by LPA which can be employed to detect LPA. Under the optimal conditions, a linear range 3.63 × 10-8-9.68 × 10-6 mol/L and a detection limit 1.27 × 10-8 mol/L of LPA were obtained. The relative standard deviation (R.S.D) was 1.8% obtained from a series of11 standards each containing 6.05 × 10-6 mol/L oLPA. This paper also discusses the mechanism of fluorescence indicator probe.