Oral mucositis, a painful and debilitating ulcerative wound condition, is a frequently occurring complication following chemo- and/or radiotherapy. While the current standards of therapy (e.g., gels and mouth rinses) provide temporary relief, there is still an unmet need for a robust, long acting barrier that can provide lubricating protection in oral wounds, thereby enhancing the wound healing response. It is proposed that an affinity based layer-by-layer (LBL) self-assembly that can be administered as a series of mouth rinses could permit the formation of protective barriers, providing a modular approach to regenerative oral therapy. In this study, biotinylated poly(acrylic acid) was synthesized for developing LBL assemblies using biotin-streptavidin affinity linkages. To explore the ability of developed LBL assemblies to potentially resist the harsh intraoral environment, in vitro chemical and ex vivo mechanical tests were performed. The stability results demonstrated significant LBL barrier stability with wear resistance. From statistical analyses, it was deduced that polymer MW and the number of LBL layers contributed significantly to chemical barrier stability. Also, the extent of biotin conjugation played a key role for LBL development and in mechanical barrier stability. Thus, the proposed affinity based LBLs with their excellent barrier properties offer a modular treatment approach in oral mucosal injuries.