PROBLEM TO BE SOLVED: To provide a membrane separation device which enables the membrane separation of a substance in a micro-fluid device and enables high-degree separation even in a separation system due to the a difference in a permeation speed or a system low in the separation capacity of a separation membrane, and the membrane separation method. ; SOLUTION: The membrane separation device for mutually separating at least two substances contained in a fluid has a inflow port for allowing the fluid to flow in and is constituted so that a first separation membrane and a second separation membrane mutually different in the separation characteristics of first and second substances are respectively provided to a flow channel wall. This membrane separation device has at least one separation unit composed of a flow channel on a supply side characterized in that the average distance between the membrane surfaces of the first and second separation membranes is 1-200 [mu]m, the first flow channel on a permeation side joined to the flow channel 1 on the supply side through the first separation membrane and having a first outflow port for allowing the fluid permeated through the first separation membrane to flow out and a second flow channel on the permeation side joined to the flow channel on the supply side through the second separation membrane and having a second outflow port for allowing the fluid permeated through the second separation membrane to flow out. ; COPYRIGHT: (C)2006,JPO&NCIPI