Objective To investigate the preparing procedures for the larger chemically acellular nerve allografts (CANA) and to establish an evaluation criteria and the preparation method.Methods The sciatic nerves ofthe dogs were exposed by a muscle-splitting incision and were isolated free of the underlying fascia. The 50-mm-long segments of the nerve were made. The proximal and distal ends of the nerve segments were labelled and stabilized by pinning the ends to a thin plastic support, and then they were treated according to the following decellularization processes: The nerve segments were rinsed with the distilled water for 9h, transferred in a 3% Triton-100 solution for 12 h, soaked in 7% sodium deoxycholate for 12 h, and washed in the distilled water for 6 h. All the decellularization steps were performed at room temperature. The nerve segments were divided into 5 subgroups. In Group Ⅰ, Group Ⅱ and Group Ⅲ, the nerve segments were decellularized for 2, 3 and 4 times, respectively. In Group Ⅳ and Group Ⅴ, the two ends of the nerve segments were ligated with a silk line and were decellularized for 2 and 3 times, respectively. Each nerve segment was subdivided into 5 portions from the proximal end to the distal end. The degrees of decellularization, activity of Laminin, degrees of demyelination, and integrity of the nerve fiber tube were observed under microscope and were assessed by a scoring system. Results In all the groups the activity of Laminin was present and the degrees of decellularization were complete. As for the demyelination of the nerve segments, the myelin sheath in Groups Ⅰ, Ⅱ and Ⅲ was partially preserved, but it completely disappeared in Groups Ⅳ and Ⅴ. The structure of the nerve fiber tube in Groups Ⅰ and Ⅳ was almost normal. The total score for the degrees of decellularization, demyelination, and structural integrity was the lowest in Group Ⅳ but the quality was the best. Conclusion The degrees of demyelination are not parallel to the times of decellularization processes. In the quality control of CANA, we should consider the following 4 factors: activity of Laminin, degrees of decellularization, demyelination, and structural integrity. For the larger CANA,ligation of the two ends of the nerve segments during the decellularization procedure may be a better method of ensuring the quality of the decellularization.