Periodontitis is a chronic inflammatory disease that can destroy periodontal tissue. Guided tissue regeneration (GTR) is widely applied to treat periodontitis. However, the challenge is to develop a GTR membrane capable of simultaneously regenerating periodontal tissue and preventing epithelial downgrowth into the defect. Herein, blended hydrogels composed of polyvinyl alcohol (PVA) and fish collagen (Col) were prepared as GTR membranes. The morphology, collagen release kinetics, and cellular behavior of the blended hydrogels were evaluated. The results showed that the surface porosity and collagen release of the PVA/Col blended hydrogels were enhanced by increasing the Col concentration. The adhesion and proliferation of human periodontal ligament fibroblasts (HPDLFs) and human gingival fibroblasts (HGFs) on the PVA/Col blended hydrogels can be regulated by tuning the PVA/Col ratio. The PVA/Col (50:50) blended hydrogel exhibited the highest cell proliferation rate for HPDLFs with spread cell morphology; the lowest viability for HGFs was found on the PVA/Col (100:0) hydrogel. Thus, by controlling the ratio of PVA to Col, multifunctional PVA/Col blended hydrogels can regulate the cellular behavior of HPDLFs and HGFs, and can be developed as GTR membranes, demonstrating their potential for guiding periodontal tissue regeneration.