In order to develop a benthic index of biotic integrity for Lake Taihu (LTB-IBI), we seasonally carried out investigation on macroinvertebrate assemblages and their environments at 32 sites in Lake Taihu from spring, 2010 to autumn, 2012. Based on the investigation, we used the method of least disturbed condition and four criteria to define reference sites. LTB-IBI was developed separately for non-central region and central region of Lake Taihu. Metrics were stepwise examined for distribution analysis, discriminating power analysis, relationship with environmental variables and redundancy analysis. The core metrics of LTB-IBI for non-central region were total taxa richness, Simpson diversity index, 3 dominant taxon% and BMWP score. One more metric, i. e. (Crustacea + Mollusca) taxa for central region. LTB-IBI value for non-central region and central region were respectively obtained by summing up core metrics, which were transformed into a uniform score by ratio score method. Health condition of Lake Taihu was found to improve gradually from 2010 to 2012. Total nitrogen was suggested as the major factor affected LTB-IBI. Our results suggest that long-term data would benefit the development of a robust LTB-IBI and its use in practice of Lake Taihu health assessment.