The oxidation of triphenylamine-4-sulphonic acid (TPASA) by hydrogen peroxide in presence of horseradish peroxidase (HRP) in weak acid medium and after incorporation of TPASA in polymeric hydrogel matrix of polyurethane (type D4) was investigated by spectrophotometry. The stability of TPASA product enzymatic oxidation under variation of reaction condition (reagents concentration, acidity) was studied. The optimum condition was selected and the possibility of HRP detection in sensor plate was shown with limit of detection 0.3•10-4 U/mL.