In this work, we have developed a carbon nanotube|Ni(cyclam)-coated glassy carbon electrode to achieve minimal fouling effects and to catalyse the oxidation of the oestrogen, estradiol, during voltammetric detection. This electrode was fabricated by initially applying a Nafion–carbon nanotube mixture, and then electropolymerising Ni(cyclam) complexes on the electrode. During this process, a two-level factorial design was used to optimise experimental parameters including the amount of carbon nanotubes, the concentration of Nafion and the surface coverage of Ni(cyclam). A linear calibration plot between 0.5 and 40 μM estradiol was then obtained in synthetic laboratory standard solutions. Based on a signal-to-noise ratio of 3, a detection limit of 60 nM was estimated, which is below the typical estradiol level measured in a normal menstrual cycle. The electrodes were subsequently applied to the detection of estradiol in protein-free human serum samples. Comparable sensitivity between synthetic laboratory standard solutions and serum samples was obtained, indicating minimal interference effects from the serum matrix.