In pH 1.8 ∼ 2.8 weak acid medium, polyvinylpyrrolidone (PVP) and Eosin Y reacted to form complex that could result in Eosin Y (EY) fluorescence quenching. The maximum quenching wavelength was at 542 nm. The fluorescence quenching (ΔF) was proportional to the concentration of polyvinylpyrrolidone in a certain range. The linear range, the correlation coefficient and the detection limit were 0.33 ∼ 2.0 μg•mL⁻¹, 0.9994 and 99.6 ng•mL⁻¹, respectively. The influences of the coexistence substances were tested and the results showed that the method had good selectivity. Therefore, a new method based on fluorescence quenching of eosin Y by PVP for the determination of trace PVP was developed. The method was sensitive, simple and rapid, which was applied to the determination of trace PVP in the beer with satisfactory results. The reaction mechanism was also discussed.