Polyelectrolyte multilayers (PEMs) have received significant attention across various fields, including biomedical, environmental, and food packaging, due to their cost-effectiveness, versatility, and accessibility. Utilizing the Layer-by-Layer approach (LbL), PEMs can be fabricated to coat solid surfaces like glass, metals, polymers, and composites. The interaction between polyelectrolytes, such as polysaccharides, proteins, amino-functionalized tannins, synthetic polymers, or their composites, often results in the assembly of PEMs in aqueous solutions with adjusted pH and ionic strength. This review presents the primary treatment methods for PEM assembly for substrates, encompassing plasma, chemical, and ultraviolet radiation treatments. The review predominantly focuses on macromolecules from renewable sources, particularly polysaccharides containing ionizable groups in their chains, such as carboxylate, sulfate, and protonated amine, employed in PEM fabrication. Durable PEMs physically stabilized by intra- and intermolecular interactions can be developed by selecting an appropriate strategy for substrate oxidation, following the selection and deposition of polyelectrolytes on the modified substrate surface. The substrate coated with the PEMs assumes specific physicochemical characteristics of the applied polyelectrolytes, such as antiadhesive, antimicrobial, anticoagulant, and binding capacity toward toxic metals or other pollutants. This review also explores PEM fabrication strategies, including conventional dipping, spray, and spin coatings, leading to the assembly of polyelectrolytes for diverse applications. This review also discusses the application of PEMs in various systems, such as drug delivery systems, scaffolds for wound healing, wound dressings, sensors, food packaging, and environmental remediation.