Abstract Background Glucose biosensors based on enzyme reaction of glucose oxidase were studied because the symptomatic therapy of diabetes mellitus requires reliable assessment of blood glucose level at frequent intervals. Tomato skin membranes have been successfully employed to entrap glucose oxidase for fabrication of glucose biosensor. Methods Glucose oxidase was immobilized onto the tomato skin and the enzyme membrane was then positioned on the surface of an oxygen electrode. The glucose concentration was quantified by the change of dissolved oxygen. All the serum samples were also simultaneously determined by a Hitachi 7060 chemistry analyzer. Results The response of the biosensor showed a linear relationship with a concentration range of 1.0–30.0 mmol/l glucose. The limit of detection was 0.20 mmol/l. Error Grid analysis demonstrated that 100% of the results fell within clinically acceptable zones A and B. The F- and t-tests showed no significant differences between the 2 methods. The recovery was 95.0–110.0% for 30 serum samples analysis. Conclusions The tomato skin biosensor possesses the advantages of simple fabrication, fast response time, low cost and high sensitivity. The results of our method are more accurate than and match well with the current clinical instrument method.