Abstract: Three-dimensional excitation-emission matrix fluorescence spectroscopy (3DEEM) was applied to measure the fluorescence properties of natural organic matter in Zhuozishan Mine groundwater. The results showed that DOM was biological source in Zhuozishan Mine surface water and groundwater (f_450/500>1.89). The detection values of TOC and UV₂₅₄ were 0.67~0.69 mg/L and 0.018~0.022 cm^-1 respectively in overlying water (including surface water and quaternary water), and the values were higher than that in sandstone water (TOC=0.24~0.62 mg/L and UV₂₅₄=0~0.019 cm^-1) and Ordovician limestone water (TOC=0.22~0.35 mg/L and UV₂₅₄=0.001~0.006 cm^-1). There were 5 fluorescence peaks of DOM in overlying water, and the fluorescence intensities (FI) of RegionⅡ, RegionⅢ and RegionⅣwere obvious. The deeper of aquifers, the fewer of quantities and intensities of fluorescence peaks decreased. There were three fluorescence peaks, and the FIs of RegionⅠ and RegionⅡ were higher than that in overlying water. The mainly fluorescence peaks were RegionⅡ and RegionⅣ, and the FIs were higher than that in sandstone water. There was a negative correlativity between the FIs of RegionⅡ and TOC, but the FIs of RegionⅣ was positively associated with TOC and the FIs of RegionⅡ. 3DEEM could be used to differentiate the fluorescence properties of DOM in each aquifer, and the results could be used for the discrimination of water bursting source.