When the in vitro research works of biosensing begin to mimic in vivo conditions, some certain three-dimensional (3D) structures of biosensors are needed to accommodate biomolecules, bacteria or even cells to resemble the in vivo 3D environment. To meet this end, a novel method of synthesizing CuO nanoflowers on the 3D graphene foam (GF) was first demonstrated. The 3DGF/CuO nanoflowers composite was used as a monolithic free-standing 3D biosensor for electrochemical detection of ascorbic acid (AA). The 3D conductive structure of the GF is favorable for current collection, mass transport and loading bioactive chemicals. And CuO nanoflowers further increase the active surface area and catalyze the redox of AA. Thus, all these features endows 3DGF/CuO composite with outstanding biosensing properties such as an ultrahigh sensitivity of 2.06mAmM−1cm−2 to AA at 3s response time. •The synthesis of 3D graphene foam (GF)/CuO nanoflowers composite was proposed.•The 3DGF provides vast area and a well-organized conducting freestanding network.•CuO nanoflowers maximize the surface area, transfer electrons between 3DGF and AA.•3DGF/CuO has an ultrahigh sensitivity of 2.06mAmM−1cm−2 to AA.•3DGF/CuO could be a smart sensing platform to support cells and detect analytes.