Abstract: A separating apparatus of biosubstance and a separating method of the same are provided. The separating method includes: providing a controller to select a positive separation process or a negative separation process to execute according to types of target biosubstances; providing a pipette pump to inject a first magnetic bead reagent or a second magnetic bead reagent into a sample according to the selected separation process, such that first immunomagnetic beads of the first magnetic bead reagent or second immunomagnetic beads of the second magnetic bead reagent are used for binding to the target biosubstances; providing a magnetic separation rack to enrich the target biosubstances so as to separate the target biosubstances and non-target biosubstances; and providing the pipette pump to separate the target biosubstances and the non-target biosubstances.