To investigate tissue culture conditions to increase certain medicinal ingredients in tissue culture seedlings of Anoectochilus formosanus, using its tissue culture seedlings as the material, set four sucrose concentrations 1%, 2%, 3%, 4% and four treatments of subculture 10, 30, 50, 70 days, and then measured the content of polysaccharide and total alkaloids from its tissue culture seedlings after culturing for some time. The results showed that 4% of sucrose concentration was the most suitable quantity on the accumulation of polysaccharide. Polysaccharide content showed an increase after the first reduction with subculture days. Total alkaloid content decreased with the increase of sucrose concentration, and it was the highest with 1% of sucrose concentration. The content of total alkaloids was the highest after 50 days of subculture.