Fungi not only play important roles in biogeochemical processes but also can form biofilm on plastic debris. However, knowledge of structure composition and spatiotemporal pattern of fungal plastisphere on different kinds of plastic debris in river with specific usages, known as river functional zones, is still missing. In this study, we investigated the spatial distribution of the fungal plastisphere across a complete urban river with different functional zones (drinking, farm irrigation, aquaculture, and tail lake). Our research was performed based on both field residual plastic debris collection and a 30-day field in situ incubation experiments. Our study revealed that plastic debris enriched distinct fungal communities (including pathogenic fungi) significantly different from the surrounding water. Tracking the source of the fungi colonized on plastic debris suggested that the fungal taxa colonized on the different kinds of plastic debris were not from the surrounding water. Human activities had considerable effects on the fungal community structure on plastic debris, and the plastisphere fungal community structure strikingly varied across different river functional zones. Plastisphere may be used as an indicator for fungi biogeography and pathogenic fungi pollution in river with different functional zones. These findings are essential for ecological risk assessment and management decisions for pollution control of plastic debris and maintaining ecological health.