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A Lake Multi-biotic Integrity Index (LMII) for the China’s second largest interior lake (Dongting Lake) was developed to assess the water quality status using algal and macroinvertebrate metrics. Algae and benthic macroinvertebrate assemblages were sampled at 10 sections across 3 subregions of Dongting Lake. We used a stepwise process to evaluate properties of candidate metrics and selected ten for the LMII: Pampean diatom index, diatom quotient, trophic diatom index, relative abundance diatoms, Margalef index of algae, percent sensitive diatoms, % facultative individuals, % Chironomidae individuals, % predators individuals, and total number of macroinvertebrate taxa. We then tested the accuracy and feasibility of the LMII by comparing the correlation with physical-chemical parameters. Evaluation of the LMII showed that it discriminated well between reference and impaired sections and was strongly related to the major chemical and physical stressors (r = 0.766, P<0.001). The re-scored results from the 10 sections showed that the water quality of western Dongting Lake was good, while that of southern Dongting Lake was relatively good and whereas that of eastern Dongting Lake was poor. The discriminatory biocriteria of the LMII are suitable for the assessment of the water quality of Dongting Lake. Additionally, more metrics belonging to habitat, hydrology, physics and chemistry should be considered into the LMII, so as to establish comprehensive assessment system which can reflect the community structure of aquatic organisms, physical and chemical characteristics of water environment, human activities, and so on.