A novel flow injection analysis (FIA) system suitable for measurements of glucose in blood serum is developed. In the proposed FIA system, a new kind of glucose sensor based on composite polymer films and well-immobilized enzyme was fabricated. An electrochemical technique of scanning electrochemical microscopy (SECM), and electrochemical impedance spectroscopy (EIS) were used for the characterization of the newly fabricated biosensor. A wide linear range of 0.1-50 mM for glucose detection was reported in virtue of the new configuration of the sensor and the developed FIA system. The reproducibility of signals was quite good with relative standard deviation (RSD) values for n = 4 injections (typically 5.7%). Animal blood serum was directly injected and assayed in this simulative physiological system. Good analytical recovery of glucose spiked into serum samples, with recoveries in the range of 96.7-105.0%, was exhibited. Under optimized conditions, detection of serum glucose for normal people and diabetics using our proposed method is possible. (C) 2012 Elsevier Ltd. All rights reserved.