The fluorescence quenching agents was characterized with three-dimensional fluorescence and ultraviolet (UV) spectra. When there was Fe (Ⅲ) in the sample, the humic fluorescence would be quenched and their UV spectra were not affected. The variation of fluorescence intensity (I) at Ex/Em=300/510　nm and UV absorbance(A) at UV300 were investigated in the article. The smaller the ratio of fluorescence intensity versus UV absorbance (I/A) is, the higher the fluorescence quencher Fe(Ⅲ) concentration is. According to Stern-Volmer equation I/I0=1－fc×Kc×［c］ /(1+Kc×［c］ ) and fitted function I/A=f×［k/(cFe3++c)+b］ , the fitted fluorescent quenching constant Kc was ranged between 1.08 to 1.15, the ratio of bounded fluorophores versus total fluorophores, i.e. fc, was ranged between 1.10 to 1.14. The ratio of fluorescence intensity and absorbance of humic acid was fitted with Fe(Ⅲ) concentration and the constants were acquired as following: f=0.83～1.19, k=587.19～612.19, c=0.87～0.92, b=－87.09～－46.36. The correlation curve values were 0.99. The Stern-Volmer formula was used to describe the quenching effect of humic acid fluorescence by Fe (Ⅲ).　However, due to the fact that the fluorescence intensity I0 without quencher was difficult to acquire during the analysis of practical samples, the fitted function between the ratio of I/A and Fe(Ⅲ) was used to reflect the quenching effect of Fe(Ⅲ) on the fluorescence of humic acid, which was based on the correlations between the fluorescence intensity I0 and ultraviolet absorbance A. The fitted formula was used to predict the iron ions concentration of the resin separated and concentrated samples from wastewater treatment plant and receiving waters. The predicted values were in good accordance with those determined with inductively coupled plasma atomic emission spectroscopy(ICP-AES) method when the iron ion concentration was above 0.4 mg·L-1, which could be used to ascertain the existence of fluorescence quenching agent and their corresponding concentration.