Separation and purification of transgenic cyanobacteria has became an indispensable step in the research of its physiology, biochemistry, molecular biology, nutritional value, etc. However, has been were no research on the separation and purification of transgenic cyanobacteria under the circumstance of cross contamination during the production of transgenic product. The method used in this article separated and purified Anabaena at three levels:species, cell and gene. In this study, the separation and purification between wild type, transgenic type and empty carrier type began at biochemical level via different culture solutions with antibiotics. Then, RT-qPCR and Western blot were applied to distinguish transgenic type and empty carrier type at molecular level. Our study not only can separate three substrain, but also obtain a result of product increase and higher growth rate. This study provides a solution to the cross contamination during the production of transgenic product and can be positive to the development of transgenic microalgae industry.