The invention discloses a free-flow electrophoresis separation chamber device used in the technical field of biochemical engineering. The device comprises an organic glass flat plate, a ceramic flat plate, an ionic membrane, an electrophoresis chamber, an electrode chamber, a fastening sealed structure and an automatic semiconductor refrigeration and heat dissipation device, wherein the ionic membrane is located between an upper organic glass flat plate and a lower ceramic flat plate, and the middle and two sides of the ionic membrane are hollowed, and the ionic membrane, the upper organic glass flat plate and the lower ceramic flat plate form the electrophoresis chamber and the electrode chamber; a plurality of liquid inlet holes are uniformly formed on one end of the upper organic glass flat plate, and double amount of liquid outlet holes are uniformly formed on the other end of the upper organic glass flat plate, thus forming a swimming lane. The device disclosed by the invention can effectively solve the problem that a separation chamber is easy to leak, can effectively eliminate joule heat in the separation chamber, can improve the stability of flowing shape of the fluid in the separation chamber, and is easy to assemble; the device can be widely used for routine free-flow electrophoresis separation of multiple types of samples such as small-molecule substances, protein, organelle and virus, and meanwhile, the device can be applied to purification of samples.