In this study we comprehensively assessed Collembola biodiversity at Three Gorges Area, one of most diverse habitat regions in China. In total, 3,796 Collembola specimens comprising 9 families, 45 genera, and 103 species (including 14 new species and new records in China) were collected from three primary sampling areas: one nature reserve and two rural and urban areas representing different degrees of human influence. Each sampling area was further divided into nine sampling sites associated with different habitats and altitudes. Analyses of biodiversity data showed that individual abundance was highest in the nature reserve followed by mildly human influenced areas, and then highly influenced areas, and species richness was lowest in highly influenced areas. Hence, we suggest Collembola biodiversity is systematically lost after urbanization. In the nature reserve, altitude significantly influenced both the species richness and individual abundance, whereas in rural and urban areas, both altitude and the human-altered environmental gradient were influential We also measured sampling efficiency and estimated potential species richness in these areas. This study serves as both a fundamental survey of Collembola biodiversity, as well as an assessment of human/environmental influence on the Collembola community, and can provide further insight into protecting the soil integrity of the Three Gorges Area.