The invention discloses a pneumatic self-circulation animal cell culture bioreactor and an application method thereof. The bioreactor is characterized by comprising a large tank, a small tank located in the large tank, a magnetic stirring system located at the bottom of the large tank, a heating system and a controlling system, wherein a sealing cover is arranged at the upper end of the large tank, jackets connected with the heating system are arranged on two sides of the large tank, and a highest liquid level sensor of the large tank is arranged at the upper part in the large tank; and a sealing head is arranged at the upper end of the small tank, an opening is formed in the lower end, the sealing head is fixedly connected in a sealing manner with the sealing cover through a breathing pipe, the upper end of the breathing pipe penetrates out of the sealing cover and is connected with a vacuum air pump and an air pressure pump respectively, the lower end penetrates out of the sealing head and enters the small tank, a cavity used for accommodating a cell carrier is defined by two up-and-down horizontal stainless steel wire mesh boards in the middle of the small tank and the inner wall of the small tank, an upper liquid level sensor of the small tank is arranged above the cavity, and a lower liquid level sensor of the small tank is arranged below the cavity. The pneumatic self-circulation animal cell culture bioreactor has the advantages of good air tightness, simplicity in operation, low contamination rate, good cell quality and high yield.