Since many chemical/biochemical reactions containing hydrogen peroxide are performed in organic solvent media, the development of a stabilized biosensor in organic solvent media is very crucial. A stable hydrogen peroxide sensor with a wide measurement range and a long life in organic solvent as well as aqueous solution was developed. To maintain the stability of the sensor in the organic solvent system, catalase was mixed with polyethylene glycol (PEG). The treatment could apparently enhance the stability of the enzyme activity. The induction of hydrogen bonding between enzyme and PEG was assumed to be the possible reason for the stabilization, and was also confirmed by infrared spectrophotometry and circular dichroism (CD). The stability of the enzyme depended upon the content and molecular weight of PEG. PEGs (MW 3,350-6,000) with a mixing ratio of 0.2 g PEG to 2.8 x 10(4) catalase activity units showed the highest stability level. The biosensor developed in the present study, therefore, worked well even in 50% (v/v) dioxane solution for 2 days; 90% of the initial activity was maintained. The detection limit of the sensor was about 140 mM and the response time was 40 s in aqueous buffer and 60-90 s in the organic solvent.