A highly-sensitive enzymatic biosensor is successfully experimented for detection of abiraterone in human serum with significance in personalized medicine and point-of-care chemotherapy of patients with metastatic prostate cancer. The dynamic range and limit of the detection of the proposed biosensor coincides with the therapeutic range of abiraterone in circulatory system of patients (below 1 μM). An optimized label-free electrochemical biosensor was exploited in order to improve the performance of biosensor to detect low concentrations of abiraterone in human serum. Electroactive surface area has been increased by 4314 mm2 by MWCNTs nanostructuring respect to bare electrode to enhance the sensitivity. CYP3A4 protein was immobilized on MWCNTs as probe biomolecule. Electrochemical cyclic voltammetries demonstrated an inhibition effect on the CYP3A4, clearly observed as a diminished electrocatalytic activity of the enzyme. Dose-response behavior of biosensor in interaction with abiraterone in human serum samples is demonstrated that shows a dynamic range between zero and 1 μM and a detection limit of 230 nM.