Ethanol is widely consumed and has been associated with various diseases in different organs. It is therefore important to study ethanol-induced responses in living organisms with the capability to address specific organs in an integrative manner. Here, we developed an autonomous system based on a series of microfluidic chips for cross-organ investigation of ethanol-induced acute response in behaving larval zebrafish. This system enabled high-throughput, gel-free, and anesthetic-free manipulation of larvae, and thus allowed real-time observation of behavioral responses, and associated physiological changes at cellular resolution within specific organs in response to acute ethanol stimuli, which would otherwise be impossible by using traditional methods for larva immobilization and orientation. Specifically, three types of chips ("motion," "lateral," and "dorsal"), based on a simple hydrodynamic design, were used to perform analysis in animal behavior, cardiac, and brain physiology, respectively. We found that ethanol affected larval zebrafish in a dose-dependent manner. The motor function of different body parts was significantly modulated by ethanol treatment, especially at a high dose of 3%. These behavioral changes were temporally associated with a slow-down of heart-beating and a stereotyped activation of certain brain regions. As we demonstrated in this proof-of-concept study, this versatile Fish-on-Chip platform could potentially be adopted for systematic cross-organ investigations involving chemical or genetic manipulations in zebrafish model.