Cyclic voltammetry was used for modification of a glassy carbon electrode with uniform worm-like cobalt oxide nanostructures (CoOx-NSt) with diameter 20–30 nm and length 100–300 nm. The electrochemical properties and morphological characterization of the modified electrode were characterized by cyclic voltammetry, SEM and AFM. The modified electrode showed a well-defined redox couple and excellent electrocatalytic activity toward guanine oxidation in a wide pH range, 5–13. The modified electrode was used as a novel amperometric sensor for guanine determination in the nanomolar–micromolar concentration range. The detection limit, sensitivity and linear calibration range of the sensor are 3 nM, 22.3 nA nM⁻¹ cm⁻² and 40 nM–10 μM, using amperommetry. Simplicity of fabrication, excellent electrocatalytic ability, high stability and fast response time are other advantages of the designed sensor. Furthermore, antifouling properties of novel cobalt oxide nanostructures toward guanine and its oxidation products improved reproducibility and repeatability of the proposed sensor.