A cholesterol biosensor of ChOx/MWCNTs containing multiwall carbon nanotubes (MWCNTs) and cholesterol oxidase (ChOx) has been successfully synthesized on glassy carbon electrode (GCE). The presence of MWCNTs not only enhances the surface coverage (Γ) but also exhibits a promising enhanced electrocatalytic activity to hydrogen peroxide. It is stable, pH-dependent, and electroactive as produced on electrode surface. The average surface coverage (Γ) was estimated about 1.86×10-11 mol cm-2 higher than both of MWCNTs and ChOx modified electrodes. Using the equation Ep = K – 2.303(RT/nF)logv and the two electrons transferred for ChOx a charge transfer coefficient 0.22 was obtained. An apparent surface electron transfer rate constant (Ks) 0.0269 s-1 was estimated for the redox peaks. Compared with the SEM images of the MWCNTs/GCE and ChOx/GCE, the ChOx/MWCNTs film shows the significant surface morphology. The electrocatalytic responses of ChOx/MWCNTs were investigated with various analytes by cyclic voltammetry (CV) and amperometry (AMP). Compared with bare electrode and ChOx/GCE, the ChOx/MWCNTs/GCE has better electrocatalytic current response to cholesterol. This sensor shows excellent performance with a sensitivity of 1261.74 μA mM-1 cm-2, a linear range of 4.68×10-5 – 2.79×10-4 M, and a detection limit of 4.68×10-5 M.