Background: Urban river pollution risks to environments and human health are emerging as a serious concern worldwide. With the aim to achieve the health of urban river ecosystem, numerous monitoring programs have been implemented to investigate the spectral characteristics of contamination. While due to the complexity of aquatic pollutants, the linkages between harmful effects and the spectral characteristics of contamination are still a major challenge for capturing main threats to urban aquatic environments. To establish these linkages, surface water (SW), sediment pore water (SDPW), and riparian soil pore water (SPW) were collected from five sites of the seriously polluted Qingshui Stream, China. The water-dissolved organic carbon (DOC), total nitrogen (TN), total phosphate (TP), fluorescence excitation-emission matrix, and specific ultraviolet absorbance were applied to analyze the spectral characteristics of urban river contamination. The Photobacterium phosphorem 502 was used to test the acute toxicity of the samples. Finally, the correlations between acute toxicity and concentrations of DOC, TN, TP, and the spectral characteristics were explored. Results: The concentrations of DOC, TN, and TP in various samples amounted from 11.41 ± 2.31 to 3844.67 ± 87.80 mg/L, from 1.96 ± 0.06 to 906.23 ± 26.01 mg/L and from 0.06 ± 0.01 to 101.00 ± 8.29 mg/L, respectively. The florescence index (FI) amounted from 1.54 to 3.14, the biological index (BIX) were between 0.94 and 1.57. The distribution patterns of specific ultraviolet absorbance at 254 nm (SUVA 254) showed that the highest aromaticity and hydrophobicity were found in SDPW and the lowest ones were in SW. All samples showed significantly inhibition on luminescent bacteria. Particularly, the highest acute toxicity was found in site 1 with an EC 50 value of 6.023-fold dilution for the raw SDPW sample. In addition, the highest fluorescence intensity was also observed from SDPW of site 1. Conclusions: Tryptophan-like and protein-like substances could be important DOC fractions contributing remarkably to the acute toxicity in the seriously polluted river. In addition, the significant reduction on acute toxicity was found with the treatment of surface water flow constructed wetland, revealing that constructed wetland could be an effective approach for toxicant degradation. These observations are useful for water treatments, and meaningful for urban sustainable development.