The invention belongs to the field of environmental microbes and relates to an isolation screening method of ammoxidation microbes. The isolation screening method comprises the following steps of carrying out enrichment culture of an isolation source in a liquid color-variable medium containing ammonia nitrogen as a single nitrogen source, carrying out color development flat and liquid culture screening to obtain a bacterial strain having an ammoxidation capability, collecting the target bacterial strain and using it for sewage treatment so that under the oxygen condition, ammonia in the sewage undergoes an ammoxidation reaction by the target bacterial strain. According to the invention, a representative aeration tank activated-sludge sample is used as the isolation source; the novel color-variable medium is adopted; the process is visible; and the isolation screening method is simple.