In this study, a sensitive and selective electrochemical sensor was fabricated by using a screen printed carbon electrode (SPCE), multi-walled carbon nanotubes (MWCNTs) and β-cyclodextrin (β-CD) for detecting cholesterol. MWCNTs were functionalized with benzoic acid moiety by employing diazonium salt chemistry, and, subsequently, a thin film of functionalized CNTs were coated on the surface of SPCE. Afterwards, β-CD was immobilized on functionalized MWCNTs modified SPCE which acts as a host to recognize guest (cholesterol) molecule specifically. Under the optimal experimental conditions and using differential pulse voltammetry (DPV) as transduction technique the sensor was able to detect cholesterol level ranges from 1 nM to 3 μM, with a detection limit of 0.5 nM. Specificity of the developed sensor towards target analyte (cholesterol) was confirmed in the presence of common interfering species including glucose, uric acid and ascorbic acid. The applicability of proposed sensor was also demonstrated for cholesterol determination in human serum samples with good recovery results (94–96%) and maximum RSD (relative standard deviation) of 4.5%.