A label-free generalized ratiometric fluorescence sensing method for quantitative analysis of Pb2+ was designed based on the interaction between the G-rich sequence AGRO100 nucleic acid probe, the fluorescence dyes thioflavin T (ThT) and N-methylporphyrin dipropionic acid IX (NMM) with the addition of Pb2+. Simultaneously, this method combined with quantitative fluorescence model (QFM) was used to analyze the concentration of Pb2+ in four different sections of Xiang River. The results showed that the generalized ratiometric fluorescent probe combined with QFM was accurate in quantitative analysis of Pb2+ in four different sections of Xiang River. The quantitative analysis results obtained by the proposed method were basically consistent with those obtained by ICP-MS analysis. The recoveries of standard addition were all in the range of 91%~108% for the concentrations of Pb2+ in different water samples.