Urban rivers affect the development and future of the city. The water quality monitoring of rivers has become an important link in environmental protection. In order to understand the water quality changes of urban rivers and the application effect of fluorescence technology in river water monitoring, three-dimensional excitation-emission matrix spectra (3DEEM) technology is used to conduct experimental analysis on a certain urban river. The results show that there are three typical fluorescent characteristic peaks in the river water. The peak A1 (λex/λem =275 nm/360 nm) is UV-type humic-like, the peak A2 (λex/λem=230 nm/350 nm) is tryptophan-like, and the peak A3 (λex/λem=275 nm/310 nm) is tyrosine-like. Besides, from upstream to downstream of the river, the protein-like fluorescent substances in the water body gradually increase, which is inferred that a small amount of domestic sewage flows in along the river. Furthermore, the fluorescence peak intensity of river water is significantly and linearly correlated with ammonia nitrogen, but has no significant correlation with COD and total phosphorus.