An optical biosensing method using enzyme-immobilized eggshell membranes for glucosinolate determination has been developed. Myrosinase-immobilized eggshell membranes were fabricated and applied to hydrolyze glucosinolates at 37 degreesC for 16 h. The glucose produced was subsequently determined by an optical glucose biosensor which was constructed with a glucose oxidase-immobilized eggshell membrane and an oxygen-sensitive optode membrane. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution. The rate change in fluorescence intensity of the oxygen-sensitive membrane was monitored and related to the glucose concentration. The effect of myrosinase loading, egg white concentration, incubation time, temperature, pH, and phosphate buffer concentration on the hydrolysis of sinigrin has been studied in detail. The myrosinase-immobilized eggshell membrane demonstrated a long shelf-life of at least 6 months and the results showed good repeatability. It has been successfully applied to determine glucosinolates contents in cruciferous and non-cruciferous vegetable samples. The results show that only cruciferous vegetables contain various amounts of glucosinolates whereas non-cruciferous vegetables do not contain any. (C) 2004 Elsevier Inc. All rights reserved.