Abstract: The Anabaena-free azolla was successfully acquired by using tissue culture from the tip of the azolla frond or eliminating Anabaena cells from inner portion of indusium cap and apical membrane of megasporo-carps. The identifications through sectioning, cavity liquid examination, N-free medium cultivation, Nase determination and scanning electromicro-scopic observation proved that the obtained azolla fronds were really Anabaena-free azolla. The Anabaena-free azolla acquired by tip culture and that acquired by removing the indusium cap and apical membrane were continuously grown in nitorgenous culture media for 9 and 2 years respectively. After that they kept making a normal growth. The fact illustrated that the above-mentioned methods to acquire Anabaena-free azolla were reliable. However, the Anabaena-free azolla obtained by chemical or physical means was unstable.The theoretical value and practical significance of Anabaena-free azo-lla were also discussed in this paper.