The gate voltage dependence of the source-drain current of a chemically assembled Fe 3 O 4 nanocrystal film single-electron transistor reflects the energy level splitting of the minority spin 3d band due to two-electron Coulomb repulsion in each nanocrystal. Using the Langmuir-Blodgett technique, a Fe 3 O 4 nanocrystal self-assembled film was deposited on a quartz substrate where a three-terminal electrode made of Au was prefabricated. At 300 K in a magnetic field of 0.2 T, the source-drain current peaked near the source-drain bias voltage of 100 V at a gate voltage of þ1 to þ3 V. At a bias voltage of 100 V, a gate voltage of þ1 V amplified the current 13 times more than at a gate voltage of 0 V. Upon increasing the gate voltage to þ5 V, the amplification of the current declined to less than three times more than at 0 V. At the optimized gate voltage, by adjusting the empty upper band energy of each Fe 3 O 4 nanocrystal over the gate electrode to match the filled lower band energy outside of the gate electrode, the tunneling current can be greatly increased, because electron transport via the empty state of each nanocrystal over the gate electrode requires no extra energy cost.