Mediatorless bienzyme-type amperometric biosensors that couple a hydrogen peroxidase (H2O2)-generating oxidase (OxD) reaction on the electrode surface with a direct electron transfer-type bioelectrocatalytic reduction of H2O2 by horseradish peroxidase (HRP) allow the detection of the substrate of the OxD without any mediator. Constructed was an analytical model involving four steps: the diffusion of the substrate of an OxD, the diffusion of oxygen, the enzymatic reaction of the OxD, and the enzymatic reaction of HRP to understand the response of the mediatorless bienzyme-type biosensors. Pyruvate oxidase was used as a model OxD to detect pyruvate. The steady-state amperometric response of the pyruvate sensor in a rotating disk mode as functions of the pyruvate concentration and the rotating speed was well explained on the model to yield the kinetic parameters for the enzymatic reactions and the mass transfer in the biosensor. Based on the kinetic parameters, we constructed a mediatorless bienzyme-type microelectrode to detect pyruvate with a steady-state diffusion-controlled performance under quiescent conditions.