Abstract: Stylonychia lemnae is a monocyte eukaryote with animal nutrition. It is also an important model protozoa to study cytology and genetics. Sterile pure culture is an important basis for the study of Stylonychia biology. In order to build a high-efficiency pure cultured system for Stylonychia, a Tetrahymena thermophila sterile pure cultured medium was referenced in this paper. And a response surface analysis was used to carried out single-factor and orthogonal experiments focused on the ratio of nitrogen source, carbon source and dipotassium hydrogen phosphate. A sterile pure cultured medium for Stylonychia was primarily established and some key cultured conditions were optimized including cultured temperature, initial pH, medium volume and initial cell density. Results showed that the securable maximum cell amount was about 3.0×10³ cells and the best conditions were initial amount 100 cells, medium volume 10 mL, initial pH 7.0 and the cultured temperature 25℃.