This study is to establish a simple and practical co-culture method of cortical neurons and astrocytes of rats. The cortex of the new-born SD rats was digested by 0.125% pancreatic enzyme, and the differential adherence was applied to obtain the mixed cell suspension of neurons and astrocytes. A low concentration of cytarabine was used to inhibit the astrocytes in a moderate way to get neuronal and astrocyte co-culture. The morphological characteristics of the cells in different times were observed under the inverted microscope. The cells began to adhere the wall 2 h after the inoculation. Neurons and astrocytes grew in a good condition under the inverted microscope 9 days after the inoculation. The results of the immunofluorescence staining and Rosenfeld's staining indicated that the co-culture of neurons and astrocytes was successful and the ratio of neurons and astrocytes was close to 1:1. A new neurons and astrocytes co-culture method, which is simple and convenient, was successfully established. It will be an efficient method for the related researches about neuronal and astrocyte co-culture in vitro.