The in vivo reconstruction of conjunctiva was investigated by using modified poly(lactide-co-glycolide) (PLGA) 50/50 scaffolds. The porous PLGA matrices were prepared by a solvent-casting particulate-leaching method with NaCl, then modified with collagen, hyaluronic acid (HA) or/and human amniotic membrane (AM) component. The growth of corneal epithelial cells and human stromal fibroblasts on the scaffolds was investigated in vitro. All the modified PLGA scaffolds demonstrated enhanced cell adhesion and proliferation as compared to PLGA untreated, and the number of cells proliferated after 1 week was increased in the order of PLGA<PLGA/collagen=PLGA/collagen/AM<PLGA/collagen/HA=PLGA/collagen/AM/HA, which was the same order as the adhesion of the cells onto the surfaces. Conjunctival wounds were created on the eyes of white rabbits bilaterally and PLGA/collagen/HA scaffolds were grafted for 4 weeks to evaluate the regeneration of ocular surface tissue at the wounds. At postoperative 4 weeks, all the wounds were completely covered with epithelial membranes. However, the contraction was minimal (6%) in the wound grafted with the modified PLGA, while that of the ungrafted wound was substantially large (25%). In addition, the collagen fibers regenerated within the scaffolds were characterized by a random array of a loose network that resembled natural conjunctiva. On the contrary, an aligned array of dense collagen fibers formed in the ungrafted wounds. Our results indicated that the modified PLGA graft as an acellular bed might allow the reconstruction of the damaged conjunctival tissue with less scar formation and contraction.