The demand for electrochemical sensors with high sensitivity and reliability, fast response, and excellent selectivity has stimulated intensive research on developing highly active nanomaterials. In this work, freestanding 3D/Co3O4 thorn-like and wire-like (nanowires) nanostructures are directly grown on a flexible carbon fiber paper (CFP) substrate by a single-step hydrothermal process without using surfactants or templates. The 3D/Co3O4 thorn-like nanostructures show higher electrochemical activity than wire-like because of their high conductivity, large specific surface areas, and mesopores on their surface. The characterization of 3D/Co3O4 nanostructures is performed by using high resolution transmission electron microscopy (HRTEM), field emission scanning electron microscopy (FESEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction analysis (XRD), and electrochemical methods. The 3D/Co3O4 thorn-like nanostructures displayed non-enzymatic higher catalytic activity towards the electrochemical detection of glucose, compared to the 3D/Co3O4 wire-like morphology. The 3D/Co3O4 thorn-like nanostructures show a wide linear range response of glucose concentration ranging from 1 to 1000 μM with a detection limit of 0.046 μM (S/N = 3). The 3D/Co3O4 thorn-like nanostructure-modified CFP electrode selectively detects glucose in the presence of 100-fold excess of interfering compounds. The 3D/Co3O4 thorn-like nanostructure-modified CFP electrode is tested with human blood serum samples and validated with commercial glucose sensors. The newly developed sensor material shows potential for glucose monitoring in clinical and food samples.