A mediator-less amperometric biosensor for putrescine detection was proposed to obtain a (pseudo) steady-state catalytic current. Putrescine oxidase (PuOD) and peroxidase (POD) were co-immobilized with glutaraldehyde on a Ketjen Black (KB)-based mesoporous electrode. A POD-catalyzed direct electron transfer-type reduction wave of H2O2 generated by the reaction of PuOD was observed at a PuOD/POD-immobilized and KB-modified rotating disk glassy carbon electrode with an onset potential of 0.60 V vs. Ag vertical bar AgCl. A PuOD/POD-immobilized and KB-modified microdisk electrode produced a spherical diffusion-controlled (pseudo) steady-state catalytic current under quiescent conditions in the presence of putrescine. The bienzyme mesoporous microelectrode exhibited a linear range from 17 mu M to 500 mu M with a sensitivity of 0.33 +/- 0.01 mA mM(-1) cm(-2) and a lower detection limit of 5 mu M (S/N > 3).