Understanding nano-bio interactions is pivotal to the safe implementation of nanotechnology for both biological and environmental applications. Zebrafish as a model organism provides unique opportunities to dissect nano-bio interactions occurring at different biological barriers. In this review, we focus on four key biological barriers, namely cell membrane, blood-brain barrier (BBB), skin and gill epithelia, and gastrointestinal tract (GIT), and highlight recent advancement achieved by using zebrafish to conduct both visualized observations and mechanistic investigations on a diversity of nano-bio interactions.