Using three-dimensional fluorescence technique for water quality monitoring has great significance to manage the river water quality in oases of arid areas. The major objectives of this study are to apply parallel factor (PARAFAC) method and self-organizing map (SOM) to assessing fluorescence properties, and to characterize and quantify the relationship between fluorescence and water quality indexes in the Ebinur Lake watershed. Fluorescence components are identified by the PARAFAC in the Ebinur Lake watershed. C1 corresponds to ultraviolet humic-like fulvic acid and C2 corresponds to humic-like fulvic acid. C3 includes two peaks, C3(T1) and C3(T2), C3(T1) corresponds to protein-like substance and C3(T2) corresponds to humic-like acid. C4 corresponds to humic-like substance. SOM technique is employed for the exploratory analysis of fluorescence components of water samples in the Ebinur Lake watershed. Discussing the distribution of water quality parameters in different clustering layers shows that the water quality tends to be better as a sequence of Bortala River upstream, JingHe Oasis, farmland surrounding Wusu, and Ebinur Lake nearby. There exist certain relationships between water quality indicators and fluorescence peaks. In wet season, the pH, electricity conductivity (EC), dissolved oxygen (DO), chemical oxygen demand (COD) and biological oxygen demand in five days (BOD5) have significant correlations with fluorescence peaks, while the total phosphor (TP), total nitrogen (TN) and NH+3-N have weak correlations with fluorescence peaks. Multiple linear regression equations are established between the pH, EC, DO, COD, BOD5 and fluorescent components, and correlation coefficients R are 0.579, 0.632, 0.502, 0.762 and 0.785, respectively. In general, based on the investigation of the fluorescence characteristics using PARAFAC, the SOM network can be used as an effective tool for analyzing water fluorescence spectra, and it can help to provide a scientific basis for water quality monitoring and river pollution controlling in arid regions.