Using Au(111) and Au(100) single-crystal electrodes modified with self-assembled monolayers (SAMs), the direct electron transfer reaction of bilirubin oxidase (BOD) adsorbed onto their surfaces was investigated. The BOD adsorbed onto the Au(111), Au(100) and gold /mica electrodes, and the BOD adsorbed onto Au(111) electrodes modified with C₃–SO₃H and C_n–COOH (n = 2, 5 and 7), showed the electrocatalytic currents of dioxygen reduction based on the direct electron transfer reaction. The BOD adsorbed onto Au(111) electrodes modified with C₆–NH₂, C₆–OH and C₅–CH₃ did not show any electrocatalytic current. Negatively charged electrode surfaces can give a suitable molecular orientation for the direct electron transfer of BOD. The k° values evaluated by an analysis of the steady-state voltammogram with a simulated fitting method did not depend on the crystal structure of the gold electrode surface. Using a C_n–COOH (n = 2, 5, 7) modified Au(111) electrode, the k° values decreased with an increasing alkyl chain length of C_n–COOH. Based on the k° values obtained from the C_n–COOH (n = 2, 5, 7) modified Au(111) electrodes, the electron tunneling distance was evaluated. The average distance between the type 1 Cu site of BOD and the outside of the BOD protein structure was evaluated to be 17 (±2) Å.