The invention provides a calibration method for eliminating quencher absorption influence in the fluorescent quenching analysis, comprising obtaining a curve (A') and a curve (B') using a practical measured spectrum, (a) of florescence generated after excitating a single fluorescent reagent by a excitation light and a practical measured spectrum (b) of fluorescence generated by a mixed sample of a fluorescent reagent and a quencher, representing unquenched fluorescent spectrum practically emitted after a direct influence absorption to the mixed sample of the excitation light and the quencher by A', representing the fluorescent spectrum of A' after quenching by B'. The method can overcome influence of the quencher absorption function in the analysis of the fluorescence quenching, making materials conventionally not suitable for analysis of the fluorescence quenching due to overlap existing in the spectrum can be analyzed by the fluorescence quenching, thus the application range of the fluorescence quenching is expanded.