The invention discloses a reciprocating type free flow isoelectric focusing electrophoresis device and a reciprocating type free flow isoelectric focusing electrophoresis method. The reciprocating type free flow isoelectric focusing electrophoresis device is mainly composed of a separation chamber, a liquid storage pipe, a gas liquid buffering chamber, collection pipes, recycling pipes which are located below the collection pipes and correspond to the collection pipes one by one, and a medium driving pump, wherein a separation chamber liquid outlet of the separation chamber is connected with the collection pipes and the recycling pipes respectively by switches; the liquid storage pipe is connected with a gas liquid buffering chamber liquid outlet; the gas liquid buffering chamber liquid outlet is connected with the separation chamber liquid outlet; and the medium driving pump drives media to flow in or out of the gas liquid buffering chamber from the gas liquid buffering chamber liquid inlet. According to the device and the method, one single-path driving pump is used for replacing a multi-channel driving pump; the rapid reciprocating of CA (Carrier Ampholytes) buffering liquid and a sample is used for replacing unidirectional circulation in the prior art by the separation chamber, so that the same effect of an existing RFFIEF technology is achieved; and the technology, equipment and method of the whole ReFFIEF technology are greatly simplified and the cost is reduced.