To provide a substance separating device capable of continuously separating two kinds of substances or more in a fluid precisely.

The separation device has a separation chamber that allows the fluid that is a sample to flow in it, a ϕ first section and a ϕ second section that are provided on a sidewall of the separation chamber and are mutually different portions of potentials that are to be driving force of separation, a first spout formed in a portion closer to the ϕ first section at a downstream end of the separation chamber, and a second spout formed in a portion closer to the ϕ second section of the separation chamber. A plurality of such separation chambers are connected over a plurality of stages.

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