Abstract: In order to make clear the hydraulic connection between upper and lower aquifers, three-dimensional excitation-emission matrix (3DEEM) was used to research fluorescent characteristics of dissolved organic matter (DOM) in each aquifer. The results showed that there were significant differences of fluorescent characteristics in each aquifer. Due to intense microbial activity in shallow part, the fluorescence intensity (FI) in region Ⅱ and region Ⅳ was high. When deep microbial activity tends to be stable, FI was very low in regionⅡand region Ⅳ. We could detect fulvic acid-like (region Ⅲ) in Quaternary aquifer, and FI of humic acid-like (region Ⅴ) was high in the lower member of Zhiluo formation. In the water sample of ZLG-8 hole, FI of aromatic protein Ⅱ was low, and soluble microbial by-product-like was not detected. These reflect deep groundwater condition and inactivity of microbe. There were same fluorescent characteristics in region Ⅴ between ZLG-8 hole and the lower member of Zhiluo formation. Overall, above analysis could make clear that groundwater of ZLG-8 hole was from the lower member of Zhiluo formation, and no direct hydraulic connection with upper aquifers.