The invention belongs to the field of fermentation engineering, and in particular relates to a method for improving the yield of amphotericin B produced through microbial fermentation. According to the method, by optimizing organic complex nitrogen sources and the fermentation conditions, the influences of the nitrogen sources of different batches on the fermentation metabolism are reduced; by adding precusor substances, the biosynthesis of the amphotericin B is promoted. After the fermentation is finished, the fermentation unit of the amphotericin B achieves 14469ug/ml. Compared with the result before optimization, the yield of the amphotericin B is improved by 103%, the fermentation time is shortened by 14%, the utilization rate of equipment is improved, and the purification of the downstream is benefited.