The macro-invertebrate assemblages of river substrate were investigated at 83 sites in Shanghai city in summer and autumn 2011. A total of 20 taxa of the macro-invertebrate were collected, including 10 of Mollusca, 6 of Arthropoda and 4 of Annelida. No any living macro-invertebrates were detected from samples collected at 9 sites which were heavily polluted and no suitable habitat available. The water quality at the rest 74 sites with living macro-invertebrate was evaluated by Shannon-Wiener index, Hilsenhoff Biotic Index (HBI) and Goodnight Biotic Index (GBI). Based on Shannon-Wiener Index, the water quality was critically polluted at 25 sites and heavily polluted at 49 sites. The water quality was heavily polluted at 38 sites, moderately at 5 sites and slightly at 31 sites, by the value of Hilsenhoff Biotic Index (HBI). Evaluating on water quality by Goodnight Biotic Index (GBI), the river water was severely polluted at 33, moderately at 2 sites, and lightly at 39 sites. In contrast with the data of historical physicochemical water quality indicators in last 3 years, Shannon-Wiener Index had low efficiency and accuracy, while Hilsenhoff Biotic Index and Goodnight Biotic Index had much higher accuracy in spatial consistency between physicochemical assessment and biological evaluation.