A new electrochemical method was designed for determination of cholesterol.by combination of continuous admittance square wave voltammetry method and a highly sensitive biosensor. The new biosensor was constructed using immobilization of cholesterol oxidase (ChOx) on multiwalled carbon nanotubes (MWCNT) and ZnO nanoparticles (ZnO). The biosensor was used in a flow injection analysis system. The special square wave voltammetric method was established based on application of fast Fourier transform (FFT) and continuously calculating the biosensor admittance in a time window. The response of the injected cholesterol samples was calculated by background subtraction and integration of the biosensor signal in the recorded voltammograms in form of coulomb. The method is very sensitive and the response time is less than 8 s. The technique was applied as an electrochemical detector in a flow injection analysis system. Detection limit of the cholesterol was 0.05 nM in a linear range from 0.2 to 60.0 nM (R-2=0.994). (C) 2013 Elsevier Ltd. All rights