The invention relates to an isolated culture method for chicken enterocyte. The method comprises the following steps: step 1, digesting the intestinal tissue block of a chicken embryo with protease so as to obtain a digestion product; step 2, filtering the digestion product with a cell strainer and collecting cell aggregate with a size of 100 to 500 meshes; and step 3, subjecting the cell aggregate to centrifugation at a speed of 800 to 1500 r/min for 5 to 15 min and collecting cell sediment. The invention further provides the chicken enterocyte obtained by using the method. The method provided by the invention can substantially shorten testing time, save reagents and improve the purity of the isolated enterocyte.