A fluid separation apparatus suitable for separating a mixed fluid with different properties and capable of separating a complex fluid mixture efficiently is provided. The fluid separation apparatus includes a sampling entrance, a first separation column, a second separation column, a bypass line, a detector and a guide multi-channel valve. A mixed fluid flows into the first separation column via the sampling entrance. The second separation column is connected to the first separation column in series and connected to the bypass line in parallel. The detector is connected to the second separation column and the bypass line. The guide multi-channel valve has different modes to control a flow-through status and a closed status between the first separation column and the second separation column, between the first separation column and the bypass line, between the second separation column and the detector, and between the bypass line and the detector.