Finding 3D biocompatible and biodegradable scaffold is important in tissue engineering which plays a critical role in transplanting methods. Several biomaterials, such as poly-l-lactide, poly(lactic-co-glycolic acid), poly(l-lactic acid)/poly(lactide-co-glycolide), alginate, collagen gel, and so on, have been applied as scaffold to culture cells in 3D environment. The most significant problem of the synthetic materials is lack of biocompatibility and bioactivity. Herein, self assemble S-layer proteins are used as a scaffold for PC12 cells culturing. For this purpose, S-layer protein was extracted from Bacillus coagulans HN68. Then, extracted S-layer was studied by SDS page and AFM. Using MTS test and Immunochemistry staining methods, the effect of self assembled S-layer scaffold on proliferation of PC12 cells was assayed. This study provides that S-layer could be an appropriate scaffold for PC12 cells culturing. Even though poly-l-ornithine is a common scaffold in PC12 cells culturing, the results show that (PLO)/S-layer is more protective.