The present invention discloses a sample preparation method of a nano-material transmission electron microscope in-situ heating chip, and the method comprises the following steps: (1) cleaning a liquid injection device; (2) connecting one end of a Peek tube with one end of a hollow glass needle tube; (3) fixing the hollow glass needle tube to a micromanipulator; (4) drawing a liquid sample with a syringe; (5) removing a liquid suction needle and connecting the other end of the Peek tube with the syringe; (6) putting an in-situ heating chip on an objective table of a microscope; (7) moving the hollow glass needle tube until the position of a tip needle corresponds to a Si3N4 film to be loaded; and (8) injecting, along the Peek tube, a nano material liquid into the hollow glass needle tube by the syringe, and dispersing the nano material liquid through the tip needle onto the Si3N4 film to be loaded. The method can accurately titrate a sample solution, simple preparation is sample, cost can be reduced, comparative experiments on one chip are facilitated, and the usage rate of the chip is improved.