An accurate and rapid analysis of human indoleamine 2,3-dioxygenase (hIDO) is crucial for the development of anticancer pharmaceuticals because of the role of hIDO in promoting tumoral immune escape. However, the conventional assay of hIDO is limited by interference from reductants, which are used to reduce the heme iron to begin the hIDO catalytic reaction. Herein, we report a direct electrochemical method to control the redox state of the heme iron and the electrochemical investigation of hIDO. Although the conventional gold electrode did not offer a visible signal for the electron transfer between the electrode and hIDO, the anodized nanostructured gold electrode exhibited a clear nonturnover electrochemical response in an Ar atmosphere. In the presence of oxygen, the bioelectrocatalytic current for the reduction of oxygen, which competes with the oxidation of tryptophan upon the addition of the substrate, was observed, confirming an electrochemically driven hIDO reaction. A well-known inhibitor of hIDO, epacadostat, hindered this catalytic signal according to its concentration, demonstrating the rapid evaluation of its inhibition activity for the hIDO reaction. Through an in silico study using the proposed electrochemical assay system, we discovered a strong inhibitor candidate with a half-maximal inhibitory concentration of 10 nM, similar to that of epacadostat. This suggests the usefulness of the proposed system in drug discovery for hIDO and kynureine pathway-targeted immunotherapy.