A novel enzymatic biosensing platform toward glucose is achieved with nanocomposite of magnetic nanoparticles (Fe3O4−CS−CD) and multi‐walled carbon nanotubes (MWCNTs). The synergistic effect of chitosan, β‐cyclodextrin and MWCNTs can facilitate electron transfer between enzyme and electrode based on the promoting results of the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The new biosensors exhibited direct electron transfer (DET) from enzyme to electrode after glucose oxidase (GOx) was immobilized on the modified electrode with the nanocomposite. Consequently, the enzymatic glucose biosensor displayed a considerably wide linear range (40 μM to 1.04 mM) with a high sensitivity of 23.59 μA mM−1cm−2, low detection limit of 19.30 μM, good selectivity, reproducibility and repeatability for detecting glucose. In addition, the current response still retained at 93.4 % after 25 days. Furthermore, the practical application of glucose biosensor was test in human serum samples with satisfactory accuracy, demonstrating promising and practical potential in biomedical diagnostics.