A biological nitrogen removal method making use of an anaerobic ammonia oxidation reaction, which comprises: causing to flow a support, under aerobic conditions, in water to be treated containing a soluble nitrogen which has been poured in a reaction tank, the support having, on the surface portion thereof, a two-layered microbial film which holds, in the outer layer, nitrite type nitrifying bacteria contributing to a nitrite type nitrification reaction or aerobic bacteria decomposing the soluble nitrogen other than ammonia into ammonia and nitrite type nitrifying bacteria contributing to a nitrite type nitrification reaction as a dominant species and, in the inner layer, anaerobic ammonia oxidizing bacteria contributing to an anaerobic ammonia oxidation reaction as a dominant species while being surrounded with the nitrite type nitrifying bacteria; and carrying out denitrification of the water to be treated by making use of the anaerobic ammonia oxidation reaction; wherein a feed rate of the support (total surface area of the support per unit capacity of the reaction tank) is adjusted to from 4 to 40 m 2 /m 3, and a flow rate of the water to be treated at the surface of the support is adjusted by stirring the water to be treated so that the maximum flow rate of the water to be treated in the reaction tank is 0.7 m/sec or greater, so that an amount of nitrous acid produced by the nitrite type nitrification reaction through the action of the nitrite type nitrifying bacteria reaches a level to inhibit a nitrate type nitrification reaction; wherein the denitrification of the water to be treated using the anaerobic ammonia oxidation reaction is performed without causing the active sludge to flow into the reaction tank; and wherein the water to be treated containing the soluble nitrogen is municipal wastewater or domestic wastewater having a soluble nitrogen concentration of 50 mg/L or less. causing to flow a support, under aerobic conditions, in water to be treated containing a soluble nitrogen which has been poured in a reaction tank, the support having, on the surface portion thereof, a two-layered microbial film which holds, in the outer layer, nitrite type nitrifying bacteria contributing to a nitrite type nitrification reaction or aerobic bacteria decomposing the soluble nitrogen other than ammonia into ammonia and nitrite type nitrifying bacteria contributing to a nitrite type nitrification reaction as a dominant species and, in the inner layer, anaerobic ammonia oxidizing bacteria contributing to an anaerobic ammonia oxidation reaction as a dominant species while being surrounded with the nitrite type nitrifying bacteria; and carrying out denitrification of the water to be treated by making use of the anaerobic ammonia oxidation reaction; wherein a feed rate of the support (total surface area of the support per unit capacity of the reaction tank) is adjusted to from 4 to 40 m 2 /m 3, and a flow rate of the water to be treated at the surface of the support is adjusted by stirring the water to be treated so that the maximum flow rate of the water to be treated in the reaction tank is 0.7 m/sec or greater, so that an amount of nitrous acid produced by the nitrite type nitrification reaction through the action of the nitrite type nitrifying bacteria reaches a level to inhibit a nitrate type nitrification reaction; wherein the denitrification of the water to be treated using the anaerobic ammonia oxidation reaction is performed without causing the active sludge to flow into the reaction tank; and wherein the water to be treated containing the soluble nitrogen is municipal wastewater or domestic wastewater having a soluble nitrogen concentration of 50 mg/L or less.