Abstract: Provided is a biosensor with a three-dimensional multi-layered structure, a method for manufacturing the biosensor, and a biosensing apparatus including the biosensor. The biosensing apparatus includes: a chamber having an inlet through which a fluid containing a biomaterial enters and an outlet through which the fluid exits; and a plurality of biosensors inserted and fixed in the chamber. Each biosensor includes: a support unit having a fluid channel through which a fluid containing a biomaterial flows; and a sensing unit disposed on the support unit in such a way that the sensing unit is exposed three-dimensionally in the fluid channel of the support unit, the sensing unit being surface-treated with a reactive material that is to react with the biomaterial flowing through the fluid channel.