As a healthy and nutritious food, the taste of honey is usually affected by glucose crystals. To investigate the glucose concentration of honey, a new electroanalytical method based on a composite material of functionalized MWCNTs with β-cyclodextrin (β-CD/MWCNTs) was hereby proposed. After glucose oxidase (GOx) loading on β-CD/MWCNTs, a new biosensor GOx/β-CD/MWCNTs/GCE was constructed for the detection of glucose in honey. Scanning electron microscopy (SEM) and infrared spectroscopy (FT-IR) proved the successful immobilization of GOx on the β-CD/MWCNTs carrier. The prepared biosensor showed a linear response to the glucose concentration in the range of 50 μM ∼ 1.15 mM with the detection limit of 0.42 μM and a sensitivity of 32.28 μA mM−1 cm−2 was obtained. The biosensor has excellent stability (95.7 % after two weeks), reproducibility (n = 10, relative standard deviation (RSD) = 2.5 %) and anti-interference. HPLC results validated the accuracy of the newly proposed method in the detection of glucose in real honey samples.