A critical observation of reconstituted collagen membrane radiated by electron beam (EB) indicated that these collagenous fibers become cross-linked network when the irradiation is carried out in greater than melt temperature and nitrogen atmosphere. Studies on the membrane properties showed that glass transformation temperature (Tg) and melt point (Tm) of reconstituted collagen have no changes, but thermal gravity curves and infrared (IR) spectra become obviously different before and after irradiation. Cross-linking density calculated by the equation based on the theory of Flory-Rehner proved further that the densities increase with radiation doses increasing. Resistance to enzymatic digestions in vitro and implantation in vivo were determined to evaluate the physicochemical properties of cross-linked matrices. Based on the above results, it was concluded that EB radiation inducing cross-linking in greater than melt temperature and nitrogen atmosphere condition is an attractive, effective method, which introduce into intermolecular covalent cross-linkings.