An amperometric enzyme electrode is described for the simple assay of rifamycin B and rifampicin. An enzyme, rifamycin B oxidase, was successfully purified from Humicola spp. To construct a enzyme electrode, a direct binding method, where a Clark electrode was coated by a solution of enzyme, BSA, and glutaraldehyde; a thin layer of cross-linked protein coating the electrode surface. The electrode can be used for assay of rifamycin B and rifampicin, without necessity for extensive separation and pretreatment. The immobilized enzyme is stable for 1 week, with small loss of enzyme activity, and exhibits a linear response to rifamycin B over the range of 0.4mM to 2.5mM and rifampicin over the range of 0.15mM to 1.0mM by maximum velocity method. The only limitation is the long response time (10 min). After preparation, the buffer is the only reagent needed.