A tiered ecological risk assessment was applied to quantitatively refine the overall probabilistic risk of cypermethrin, a pyrethroid insecticide, to aquatic organisms. These results were then validated through the bioassays using field water from an urban stream, Chebei Creek in Guangzhou, South China. Seventeen water samples were collected along Chebei Creek for evaluation. In total, 71% of the field waters were acutely toxic to Hyallela azteca and 24% of the waters caused 100% mortality. Toxic unit evaluation suggested that cypermethrin was one of the main contributors to toxicity. The tiered ecological risk assessment approach (deterministic quotient method and probabilistic methods, including joint probability curve and Monte Carlo Simulation) suggested that cypermethrin posed significant threats to aquatic ecology in this stream. The overall probabilistic risk of cypermethrin to aquatic species in Chebei Creek reached 66% when acute-to-chronic ratios were set at 125. An exceedance probability of cypermethrin in Chebei Creek that affected H. azteca as modeled using the joint probability curve method was 88%, suggesting that most sites were at risk due to cypermethrin exposure. This value was similar to the results obtained from acute toxicity tests (71% of field water samples were acutely toxic to H. azteca), indicating the effectiveness of the tiered approach to assess risk of cypermethrin in urban waterways. To the authors' knowledge, the present study is the first to provide a focused probabilistic evaluation of ecological risk for cypermethrin in a complex urban waterway environment. Despite uncertainties existing in the ecological risk assessment procedure, this approach provides a comprehensive assessment of ecological risk of cypermethrin, and subsequently, a foundation for further risk diagnosis and management in urban waterways.