The design of bimetallic selenide configurations has been regarded as a feasible strategy to modulate the electronic structure of the electrocatalysts for sensors application. In this sense, unique hierarchical porous three-dimensional Ni–Co diselenide nanowires (3D Co0.6Ni0.4Se2 NWs) coated with N-doped carbon thin layer (3D N-C@Co0.6Ni0.4Se2 NWs) with hollow sea-urchin-like overall morphology were fabricated and used as an effectual bifunctional electrocatalyst for glucose oxidation and hydrogen peroxide reduction. Regarding the advantages of this porous and 3D structure, the N-C@Co0.6Ni0.4Se2 NWs offer abundant electrochemical-active sites, high availability to the electrolyte/analyte, and high conductivity due to the synergistic effect of bi-metals, coated N-doped carbon and selenide. As an amperometric glucose sensor, it shows a high sensitivity of 1439 and 239 μA mM−1 cm−2 and wide linear range from 0.5 μM to 2.2 mM and 2.2 mM–12.2 mM with a low detection limit of 0.16 μM. Moreover, amperometric H2O2 sensing will be recognized with a sensitivity of 89 and 31.8 μA mM−1 cm−2, a linear range from 0.5 μM to 6 mM and 6 mM–15 mM, and a detection limit of 0.16 μM (S/N = 3). Moreover, the developed sensor shows a great potential in determining glucose and H2O2 in biological real samples.