Abstract: A novel enzyme of bilirubin oxidase produced by a genus Myrothecium or genus Coprinus origin microorganism and a conventional enzyme of laccase are found, in the presence of a specific additive compound, e.g. a surface active agent, aromatic carboxylic acid, sulfa drug or protease, to oxidize both conjugated and unconjugated bilirubin in biological fluid to biliverdin without formation of hydrogen peroxide, such that in the case of conventional enzymatic methods of the quantitative determination of glucose, cholesterol, neutral fats, free fatty acids, phospholipids or uric acid all existing together with bilirubin in biological fluid, the usual interference with such determination, as otherwise caused by bilirubin coexisting in such fluid, can be prevented by adding such a bilirubin oxidase or laccase together with such a specific additive compound to the determinative reaction system.