To find best methods to prepare acellular dura and sclera, and compare the effects of different methods including processes, concentrations and time. METHODS:The duramater and sclera which came from 8 New Zealand rabbits were divided into 8 groups. Each group had dura and sclera. 5, 10, 20, and 50mL/L Triton X-100 were used in 4 groups. Other 4 groups were treated with 2.5g/L trypsin and 1g/L SDS, and the treated time was 12h+12h, 12h+24h, 24h+12h and 24h+24h. After these procedures, the appearances of those tissues were observed, residual cell number was counted after HE staining, and electron microscope was used to observe the ultrastructures of dura and sclera. RESULTS:The appearances of acellular tissues treated by Triton X-100 had no obvious differences with tissues that hadnt been treated. Two groups treated by 20mL/L and 50mL/L Triton X-100 and three groups treated by 2.5g/L trypsin and 1g/L SDS with treated time≥36 hours had the least residual cells. After acellular procedures, dura and sclera had the same ultrastructure as pretreatment in electron microscope. CONCLUSION: The collagen fibres of dura and sclera treated by Triton X-100 were well conserved. The residual cells are less in tissues treated by 20mL/L Triton X-100. 20mL/L Triton X-100 is fit for treating dura and sclera.