Rivers are known as major pathways for transporting microplastics from terrestrial areas to the marine environment. However, the behavior of microplastics in terms of retention and transport within riverine systems remains unclear. While considerable efforts have been made to investigate the water column and sediment, limited attention has been given to understanding the interplay between microplastics and benthic biofilms. Therefore, this study aimed to examine the distribution of biofilm-trapped microplastics along the CaoE River and identify the factors influencing the immobilization of microplastics by benthic biofilms. The findings of this study revealed that benthic biofilms served as a sink of microplastics in the CaoE River, with an average abundance of 575 items/m2 in tributaries and 894 items/m2 in the main stream. The dominant shape of microplastics was fiber, while the primary polymer type was polyethylene terephthalate. The distribution of microplastics exhibited significant spatial heterogeneity, as indicated by their abundance and characteristics. In order to reveal the intriguing phenomenon, variations of influencing factors were estimated, including physicochemical characteristics of water, extracellular polymeric substances of benthic biofilms, and microbial communities of benthic biofilms. A partial least squares path modeling analysis was performed using these variables, revealing that water velocity and microbial diversity of benthic biofilms were the key factors influencing the interaction between microplastics and benthic biofilms. In summary, this study provides substantial evidence confirming the crucial role of benthic biofilms in the immobilization of microplastics, which expands concerns about microplastic pollution in the riverine systems. Furthermore, uncovering the underlying influences of microplastic-biofilm interactions will facilitate the development of effective strategies for the control and management of microplastic pollution.