The present invention discloses a nanometer material transmission electron microscope in-situ testing chip, a preparation method and applications thereof, and belongs to the technical field of nanometer material performance in-situ testing. The chip comprises a silicon substrate, insulating layers and a film window, wherein the insulating layer respectively grows on both surfaces of the silicon substrate, a metal film or device or a semiconductor function film or device grows on the chip front surface insulating layer so as to apply various physical and chemical effects on a sample, the center of the chip is provided with the film window, and the film window region is provided with a electron beam penetrating long hole or electron beam penetrating long groove having a large aspect ratio. According to the present invention, the transmission electron microscope sample can be subjected to in-situ measurement under the atomic scale resolution, the nano-wire samples and the nano-tube samples can be subjected to in-situ characterization, the bulk samples and the heterojunction interface samples can be subjected to in-situ characterization, the placement of the sample through the micro manipulator in the focused ion beam system and the laboratory can be achieved, and the sample transferred and fixed on the chip can be continuously processed by using the ion reduction equipment.