The invention discloses a 3D multi-layer high-flux organ chip as well as a preparation method and an application thereof, and belongs to the field of biological tissue engineering. The chip comprisesa liquid storage layer, a thin film layer and a 3D culture layer which are stacked in sequence. The method is characterized by completing bonding connection of the liquid storage layer, the thin filmlayer and the 3D culture layer by utilizing PDMS glue, and constructing a biological barrier cell layer on the thin film layer on the liquid storage layer side of the chip, and carrying out 3D cell culture in the culture micropores of the 3D culture layer of the chip to construct the biological barrier model. The chip can be used for researching interaction of two cells, angiogenesis, drug permeation, cell polarization, cell migration, drug activity evaluation and the like. Moreover, an in-vitro model and a biological barrier model which are combined with high-throughput drug screening can beconstructed, and the method is used for screening of anti-brain glioma compounds of blood-brain barriers and related research. Moreover, the liquid is convenient to operate and suitable for high flux,and external equipment is not needed. The construction method is simple and effective.