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According to the new assay for lecithin, phospholipase D (PL), choline oxidase (ChOX), and the indicator conjugate bromothymol blue−glutathione (BTB−GSH) are co-immobilized on glutaraldehyde-activated polyacrylamide transparent gel. When a sample containing lecithin is added to the biocatalytic gel, lecithin is hydrolyzed by PL to choline, which is oxidized to betaine by ChOX. The organic acid betaine changes the microenvironmental pH of the enzymes, thus leading the optical absorption of the biocatalytic gel to a hypsochromic shift because of the co-immobilized indicator conjugate, BTB−GSH. The change of the optical absorption at λmax (622 nm) is related linearly to lecithin concentration in the range 20−120 μM. When the optical effect is quantified in a spectrophotometer, the analyte concentration can be determined. The biocatalytic gel is used successfully for lecithin determinations in bovine serum and soybean granules. The principle of the method is applicable to the construction of optical biosensors. Keywords: Bromothymol blue; choline; choline assay; choline oxidase; enzyme assay; immobilized enzymes; lecithin; lecithin assay; optical biosensor; phosphatidylcholine; phospholipase D