The invention discloses an air exhausting device of a free-flow electrophoresis separation cavity and an implementation method of the air exhausting device, and belongs to the technical field of biochemical engineering. The air exhausting device comprises an air inflating pump, a hose clamp, a rubber hose, an air-liquid buffering and separation device, a self-balancing recycling device, a separation cavity shell, a separation cavity, a waste liquid recycling bottle and a recycling three-way valve. According to the air exhausting device, the liquid storage function of the air-liquid buffering and separation device is utilized, firstly, the buffering fluid which is over ten times as large as the volume of the separation cavity is stored in the air-liquid buffering and separation device, then the buffering fluid stored in the air-liquid buffering and separation device quickly enters into the separation cavity by virtue of a hand-operated air inflating pump, as the speed of the buffering fluid is high, the separation cavity can be filled with the buffering fluid soon, thus the air in the separation cavity is exhausted completely. The device disclosed by the invention can be used for exhausting the air in the separation cavity by utilizing very simple operations within a short period of time, so that the problem that the former separation cavity is cockamamie and difficult to exhaust air; and the device has the advantages of simple structure, low cost, practicability and effectiveness, and is easy to operate and the like.