More than 350M tons of plastics are produced over the world; eventually, plastic is degraded into microplastics/nanoplastics and released into the environment. Compared to the inert bulk plastics, the impacts of nanoplastics on the health are still largely underexplored. For epithelial tissues, the dynamic of tight junctions is important in maintaining the barrier integrity. In this study, using a microfluidic system, we build up a multi-layer model to reconstruct the epithelial barrier to investigate the influence of nanoplastics on the epithelial barrier. To support long-term culture and real-time monitoring, the microfluidic chip is designed with medium-exchange channels and a side-view observation window. A multi-layer cell model is established via layer-by-layer co-culture of human keratinocytes and fibroblasts representing the epidermis and dermis; the thickness of the on-chip epidermis (0.1–0.2 mm) and the on-chip dermis (0.3–0.6 mm) is close to the clinical observations. Applying nanoplastics to the epithelial barrier, we found the disassembly of tight junctions along the intercellular contacts; nanoplastics further penetrate deeply into the multicellular structure. Moreover, we observed the increasing intracellular reactive oxygen species (ROS), which suggests the potential dysfunction of mitochondria led by nanoplastics internalized into the cells. Our data suggested that the nanoplastics exposure may lead to the destruction of epithelial functions through the tight junction disassembly. Considering the critical roles of tight-junction and ROS in homeostasis and disease progression, besides the nanotoxicity, our findings suggest an alternative impact of nanoplastic on our health through interrupting the junction assembly.