An electrochemical impedance biosensor was proposed for the detection of small molecule biotin based on the plasma-polymerized films (PPF) and the bioaffinity difference between an analyte (biotin) and an analogue compound (HABA) in binding avidin. Avidin formed a metastable complex with 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) immobilized on the electrode surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The impedance spectra change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the impedance change and the biotin concentration. The proposed electrochemical impedance bioaffinity sensor has nice response to biotin in the range of 4.8×10-9–5.6×10-4 M. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.