Background Stroke and traumatic injury to the nerve system may trigger axonal destruction and the formation of scar tissue, cystic cavitations and physical gaps. Olfactory ensheathing cells (OECs) can secrete neurotrophic factors to promote neurite growth and thus act as a prime candidate for autologous transplantation. Biological scaffolds can provide a robust delivery vehicle to injured nerve tissue for neural cell transplantation strategies, owing to the porous three-dimensional structures (3D). So transplantation of the purposeful cells seeded scaffolds may be a promising method for nerve tissue repair. This study aimed to evaluate the compatibility of a novel collagen-heparan sulfate biological scaffold with olfactory ensheathing cells in vitro. Methods Collagen-heparan sulfate (CHS) biological scaffolds were made, and then the scaffolds and OECs were co-cultured in vitro. The viability of OECs was tested by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium (MU) assay at days 1, 3, 5 and 7. Statistical analysis was evaluated by student's t test. Significance was accepted at P <0.05. OECs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and the CFSE-labeled OECs were seeded into CHS scaffolds. The attachment and growth of OECs in CHS scaffolds were observed and traced directly by fluorescent microscopy and environmental scanning electron microscope (ESEM). Results CHS biological scaffolds had steady porous 3D structures and no cytotoxicity to OECs (F=0.14, P=0.9330). CHS biological scaffolds were good bridging materials for OECs attachment and proliferation, and they promoted the axonal growth. Conclusion The compatibility of CHS biological scaffolds with OECs is pretty good and CHS biological scaffold is a promising cell carrier for the implantation of OECs in nerve tissue bioengineering. Chin Med J2010;123(10):1299-1304