The aim of this study was to develop a new active coating of layer-by-layer (LbL) structure composed of alginate (as polyanions) and chitosan (as a polycation) containing sulfated polysaccharide (fucoidan) from Sargassum angustifolium, to protect rainbow trout fillets during refrigerated storage. Chitosan and alginate do not combine with each other as a homogeneous solution, so they are suitable for multilayer coatings. The results demonstrated that coating samples with chitosan and fucoidan significantly improved the quality of fish fillets and extended their shelf life from 6 to 16 days. The chemical values (TBARS and TVB-N) and bacterial growth (total viable count (TVC), total psychrophilic count (PTC), and lactic acid bacteria (LAB)) indicated lower levels in the LbL coating samples containing fucoidan compared to the alginate and control samples. Among the different coating samples, the LbL coating with fucoidan (AChF1) exhibited lower weight loss, improved chromaticity (L*, a*, and b*), and minimal changes in mechanical and sensory evaluations. Based on the findings, AChF1 was the most effective treatment for increasing the shelf life of rainbow trout fillets during refrigerated storage. Therefore, it has potential applications in the food packaging industry.