The entrapment of sulfite oxidase (SOx) into ultrathin polypyrrole (PPy) films of 27-135 nm thickness has been successfully used for amperometric biosensing of sulfite with considerably improved performance. Optimum galvanostatic entrapment was accomplished in an electrolyte-free solution which contained 0.1 M pyrrole and 5 U/mL of SOx with a polymerization period of 120 seconds and an applied current density of 0.2 mA cm-2. Evidence of the incorporation and retention of SOx in the ultrathin PPy film was obtained by scanning electron microscopy, cyclic voltammetry and amperometric measurements. Entrapment of the enzyme in a 54 nm thick PPy-SOx film gave optimum amperometric response for sulfite and enabled the detection of as little as 0.9 μM of sulfite with a linear concentration range of 0.9 to 400 μM. The successful application of the biosensor to the determination of sulfite in beer and wine samples is reported. Comparison with a spectrophotometric method indicates that the biosensor was more superior for the determination of sulfite in red wine.