Abstract: The present invention provides a detection device for body fluid separation, which comprises the plurality of bottles, the upper knob member having an upper outer casing and an upper inner casing sleeved within the upper outer casing, and the lower knob member having a lower outer casing and a lower inner casing sleeved within the lower outer casing. When the upper outer casing is tightly coupled to the upper inner casing or the lower outer casing is tightly coupled to the lower inner casing, the bottles do not communicate with each other. When the upper outer casing is separated from the upper inner casing or the lower outer casing is separated from the lower inner casing, the bottles communicate with each other. Accordingly, the detection result can be quickly determined by the detection device and detection method for body fluid separation.