A three-dimensional (3D) macroporous carbon (3D-KSCs) derived from kenaf stem (KS) is proposed as a novel supporting material for electrochemical sensing and a biosensing platform. A series of 3D-KSCs/inorganic nanocomposites such as Prussian blue (PB) nanoparticles (NPs)-carboxylic group-functionalized 3D-KSCs (PBNPs-3D-FKSCs), CuNiNPs-3D-KSCs, and CoNPs-3D-KSCs were prepared by a facile two-step route consisting of carbonization and subsequent chemical synthesis or one-step carbonization of KS-metal ion complex. The obtained 3D-KSCs/inorganic nanocomposites were characterized by X-ray diffraction, X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy, scanning electron microscopy, and Fourier transform-infrared spectroscopy. A whole piece of 3D-KSCs/nanocomposites was used to prepare an integrated 3D-KSCs/nanocomposite electrode. Compared to the electrode modified by graphene, carbon nanotubes and their derivatives, which can form close-packed structure after assembled on electrode surface, the integrated 3D-KSCs/nanocomposite electrode shows a 3D honeycomb porous structure. Such structure provides a large specific surface area, effectively supports a large number of electro-active species, and greatly enhances the mass and electron transfer. The electrochemical behaviors and electrocatalytic performances of the integrated 3D-KSCs/inorganic nanocomposite electrode were evaluated by cyclic voltammetry and the amperometric method. The resulted PBNPs-3D-FKSCs, CuNiNPs-3D-KSCs, and CoNPs-3D-KSCs electrode show good electrocatalytic performances toward the reduction of H2O2, the oxidation of glucose and amino acid, respectively. Therefore, the low-cost, renewable, and environmentally friendly 3D-KSCs should be promising supporting materials for an electrochemical sensor and biosensor.