A new choline oxidase (ChO) and toluidine blue O (TBO) based amperometric choline biosensor was reported in this article. An amperometric choline biosensor with immobilization of TBO (as a mediator), ChO onto polypyrrole-polyvinylsulphonate (PPy-PVS) film was accomplished on the surface of a platinum electrode. ChO was immobilized on PPy-PVS film by cross-linking with glutaraldehyde (GA). TBO was used as the mediator. Choline is oxidized to betaine and hydrogen peroxide in an oxygenated environment by ChO. Mediator reduced by reaction with hydrogen peroxide. The amperometric response was based upon the electrocatalytic properties of TBO. Optimum pH and temperature values were 7.0 and 30 degrees C, respectively. There was linearity between 1.0 x 10(-8) and 2.0 x 10(-8) M (R-2 = 0.9805). The detection limit of the biosensor was 1.0 x 10(-9) M and response time of the biosensor was 200 s. The storage stability and reproducibility of the biosensor were also investigated. Interfering effect of several interferants such as ascorbic acid, uric acid, alanine, dopamine, paracetamol, cysteine, and glucose on the choline biosensor was examined. The developed biosensor was tested in determinations of content in a synthetic blood sample.