Fluvial-delta plain complex links land, fresh water, and marine habitats and acts as a springboard during terrestrialization of both plants and animals. Its geological records or related paleoecology study, during mid to late Paleozoic, however, are scarce. In this study, we reconstruct the paleoenvironmental and paleoecological framework of a Middle Devonian (c. 387 Ma) fluvial-delta plain complex from Wuding, eastern Yunnan, South China for the first time, using a combined approach of sedimentology, geochemistry, and palaeontology. Eight lithofacies of fluvial-delta plain are recognized from the Haikou Formation. The lower part of the Haikou Formation deposited in a meandering fluvial environment. While the middle-upper part of the Haikou Formation is interpreted as a brackish-fresh water setting (i.e., delta plain-swamp) based on facies analysis, stable isotope composition (delta 18O, delta 13C) of biogenic (ostracods) and authigenic carbonates, as well as paleosalinity proxies (Sr/ Ba). A synthetic biota with a variety of fossil organisms, including ostracods (29 species belonging to 7 super-families), charophytes (1 species), chondrichthyans and antiarch fishes (at least 2 taxa), gastropods (1 taxa), bivalves, and spores (20 species belonging to 19 genera) have been recognized and identified in the delta plain-swamp facies. Ostracods are categorized into three distinct assemblages. Assemblage 1 (Leperditicope-Palaeocope assemblage) and Assemblage 3 (Paraparchitoidean assemblage) displaying high diversity and abundance, and characterize a brackish lower delta plain and a delta plain-swamp environment, respectively. Whilst the Assemblage 2 (Leperditioidean assemblage) has relatively low diversity and inhabited in fresh water, probably an upper delta plain setting. Leperdiditioideans are pioneers colonized fluvial-delta plain setting and might be the earliest fresh water ostracods. Additionally, a special fresh water species of Cypridoidea, Carbonita sp., which has never been reported prior to the Carboniferous Period, is discovered in the Haikou Formation. Abundant rhizomes of vascular land plants, as well as vertically arranged traces fossils (i.e., Scoyenia beerboweri) are observed from the flood plain facies, representing development of belowground soil ecosystem. The biota, especially those of the ostracods and charophytes, comprise a complex ecosystem spanning the marginal marine to terrestrial settings allowing a well-structured reconstruction of the eco-pyramid of brackish-fresh water ecosystem during the Middle Devonian. The results shed light on the co-evolution of the environment and organisms in the Devonian non-marine system.