A karst system, formed by the dissolution of carbonate rocks, is usually susceptible to contamination. Little is known about the composition of natural dissolved organic matter (DOM) in groundwater systems, especially in karstic groundwater. To reveal the characteristics of DOM in a karst aquifer, the Yufuhe River Basin, a typical karst watershed in northern China, was selected. DOM fluorescence (FDOM) was measured with the excitation-emission matrices (EEMs) spectroscopy technique. Parallel factor analysis (PARAFAC) was used to analyze the karst hydrogeological factors that affect FDOM biogeochemical behavior. Three fluorescent components, i.e., tyrosine-like, tryptophan-like, and ultraviolet fulvic acid were found. Their fluorescence properties were closely related to human activity and subterranean hydrology. Fluorescence properties suggested that FDOM in the Yufuhe River karst aquifer was predominant from anthropogenic activity. In addition, due to the effect of karstic heterogeneous hydrological conditions, FDOM showed obvious differentiation in the recharge, flow path, and discharge systems. The FDOM fluorescence intensity (FI) was weak in surface water and groundwater at the upper reaches (recharge area). In the middle of the flow path area, the percentage of tyrosine-like and tryptophan-like substances degraded and fulvic acid rose gradually. However, after infiltrating into the lower reaches (discharge area) of the deep karst aquifer system, the fulvic acid matter was consumed and protein-like matter accumulated.