Abstract It is significantly important to design enzyme-free electrocatalyst for sensitive and selective detection of glucose (Glu). In this paper, we report that metallic nickel nitride nanosheet on Ti mesh (Ni 3 N NS/Ti) behaves as an efficient three-dimensional catalyst electrode for Glu electro-oxidation under alkaline conditions. We further demonstrate the use of such Ni 3 N NS/Ti as a non-enzymatic electrochemical Glu sensor superior in analytical performances. The introduction of the Ni 3 N NS/Ti can provide a high surface area and lead to high sensitivity to Glu. This sensor shows a fast amperometric response toward Glu and can achieve steady state current density within 5 s, a detection range of 0.2 μM–1.5 mM, a detection limit of 0.06 μM (S/N = 3), a response sensitivity of 7688 μA mM −1 cm −2 , and satisfactory selectivity and reproducibility. In addition, it also works effectively for Glu detection in human blood serum, promising its application toward determination of Glu in real samples.