Intestinal epithelial cells(IECs) are important functional cells in intestine, while intestinal barrier, a complex system , is formed mostly by intestinal mucosa cells, Primary cultured intestinal epithelial cells is thus the effective cell model in research in nutrition and physiology, pathology. Ctenopharyngodon idellus is the freshwater fish with important economic and food value in China. No practice operation method and system evaluation method of primary culture intestinal epithelial cells from Ctenopharyngodon idellus are published. The aim of this study was to establish a method specification on dissociation of IECs from intestinal mucosa of Ctenopharyngodon idellus, as well as effective evaluation index of primary culture of IECs. In this study, three digestion methods and different centrifugal speeds were used on the intestinal mucosa. The combinations of three culture mediums and four concentration gradients of CO2 were tested, as well as different concentration gradients of serum and cells seeded. After observing the growth process of IECs, we choose three observation methods, MTT method and enzyme activities interrelated to system evaluate the growth effect of primary cultured IECs. The results showed that the tested fish need to be intensified rearing to protect test repeatability. Using mechanical scrape plus collagens digestion and centrifugal speeds on 400r/min, cells grew in M199 added 15% serum concentration at 27℃ in 6% CO2 incubator , while the seeding concentration was 2000 clumps in each plate hole, intestinal mucosa cells’ growth was stability. The growth process of primary cultured IECs could be evaluated system of the combinations of inverted fluorescence microscope method and Giemsa staining method, as well as MTT method, activity of AKP and ratio of LDH / MTT OD.