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, physiology and pathology. Ctenopharyngodon idellus is the freshwater fish with important economic and food value in China. No practical operation method and system evaluation method of primary culture intestinal epithelial cells from Ctenopharyngodon idellus were 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 CO2 were tested, as well as different concentration gradients of serum and cells seeded. After observing the growth process of IECs, we chose three observation methods, MTT method and enzyme activity interrelated system to 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 at 400 r/min, cells grew in M199 added 15% serum concentration at 27 ℃ in 6% CO_{2 incubator , while the concentration of cells seeded was 2 000 clumps of cells in each plate hole, intestinal mucosa cells’ growth was stable. The process of primary cultured IECs growth could be evaluated system of the combinations of inverted fluorescence microscope method and Giemsa staining method, as well as MTT method, enzyme activity of AKP and the ratio of LDH/MTT OD.}