AIM: To investigate the antifibrotic effect of freeze-dried bilayered fibrin-binding amniotic membrane on trabeculectomy in a rabbit model. METHODS: Twenty-four Japanese white rabbits were randomized into three groups: the experimental group (ocular trabeculectomy in combination with freeze-dried bilayered fibrin-binding amniotic membrane transplantation), the control group (ocular trabeculectomy in combination with natural bilayered fibrin-binding amniotic membrane) and the blank group (single trabeculectomy). Clinical observation, hematoxylin-eosin staining, Massion staining, real-time PCR and immunohistochemistry for α-SMA were performed on days 7, 14, 21 and 30 following surgery. RESULTS: Statistical differences were noted in survival analysis and intraocular pressure(IOP) among groups on days 7, 14, 21 and 30 following surgery. Histology, immunoh- istochemistry and real-time PCR further demonstrated that trabeculectomy in combination with freeze-dried bilayered fibrin-binding amniotic membrane resulted in good wound healing and no scar formation. CONCLUSION: Self-made freeze-dried bilayered fibrin- binding amniotic membrane may inhibit the formation of scarring in glaucoma after trabeculectomy.