To continuously separate two or more substances contained in a fluid by multistage separation and obtain a high yield.

The device 50 of separating substances is constituted so that two members 1, 2, on any of which a plurality of groove-shaped separation flow passages 3a or 3b are formed almost in parallel, are fixed liquid-tightly to each other so that the flow passages 3a and 3b are made to cross each other and communicate with each other to form a separation area 5. A introduction port 7 is arranged on the upstream side of the separation area 5, first and second takeout ports 8, 9 are arranged at both ends of the separation area 5 on the downstream side and a third takeout port is arranged in a portion of the separation area 5 except both ends. A temperature gradient, an electric potential gradient, a magnetic potential gradient, acceleration, ultrasonic vibration, temporally-asymmetric vibration, adsorption/desorption, chemical affinity or the like is applied in directions almost orthogonal to the respective separation flow passages as a driving force for separation, so that the first substance is separated from the fluid and collected in one of the separation flow passages 3a and 3b and the second substance is separated from the fluid and collected in the other of the separation flow passages 3a and 3b successively in the crossed parts of the separation flow passages 3a and 3b. The fluid containing the first or second substance whose concentration is increased by repeating the separation/collection is recovered from the first or second takeout port 8 or 9.