In this work, the diffusion of thiocholine ion into an enzyme-loaded polypyrrole film was evaluated by different methods, and the results were compared to identify the most suitable method. The enzyme-loaded polypyrrole film was coated with a thin layer of gelatin and gluteraldehyde so as to prevent enzyme leaching. Diffusion coefficients under normal and prepolarized conditions were calculated by five different methods, namely, the Cottrell method, the method of Peerce and Bard, the theoretical impedance model, the electrochemically stimulated conformational relaxation (ESCR) method, and the direct impedance measurement method. The theoretical model of Vortynstev was used to calculate the parameters from the impedance spectra using simplex technique in MATLAB. The results indicate that under normal unpolarized condition the ESCR method gives a diffusion coefficient close to that given by Vortynstev method, but under polarized conditions the Cottrell method can provide a better value of diffusion coefficient than ESCR. The diffusion coefficient of thiocholine in PPy composite film from an electrolytic background of phosphate buffer of pH 7.4 was found to be 1.00 x 10(-8) cm(-2) s(-1) based on Vortynstev method. The mechanism of thiocholine diffusion into the positively charged/polarized matrix is attributed to be through the formation of a dinegative ion between thiocholine and phosphate anion via electrostatic attraction.