Several studies have demonstrated the potential of olfactory ensheathing cells (OECs) for the repair of central and peripheral nerve injury. In this work, ensheathing cells that express the receptor gene coding for enhanced green fluorescent protein (eGFP) from adult mice were isolated and purified, and then, its biological characters were examined in vitro. The work was based on combinations of fluorescence confocal, phase contrast, cell proliferation assay and immunolabeling identification. The results showed that (1) two major morphologically and immunohistochemically distinct types of cells were present after 15 days in the primary cultures of adult transgenic mice olfactory nerves and glomerular layers of the olfactory bulb. One cell type was bipolar or multipolar OECs and strained positively for P75 low affinity neurotrophic receptor (P75N), S100, and glial fibrillary acidic protein(GFAP). The other type was fibroblasts with flat or endothelial-like shape, and reacted with antibody against Thy1.1. (2) A simple, inexpensive method for purifying ensheathing cells, in which various harvested cell types showed different rates of attachment to the uncoated culture ware, was developed. This technique neither binds any antibodies nor requires any costly equipment, and yields a large number of highly purified cells. (3) In sequential observations over 22 days in culture, the population of purified cells was maintained and continued to proliferate for longer. This experiment not only supported and advanced the ensheathing cell research but also offered ideal materials of in vivo transplantation for the repair of CNS injury.