The layer-by-layer (LbL) assembly technology has been widely used to functionalise surfaces and precisely engineer robust multilayered bioarchitectures with tunable structures, compositions, properties, and functions at the nanoscale by resorting to a myriad of building blocks exhibiting complementary interactions. Among them, marine-origin polysaccharides are a sustainable renewable resource for the fabrication of nanostructured biomaterials for biomedical applications owing to their wide bioavailability, biocompatibility, biodegradability, non-cytotoxicity, and non-immunogenic properties. Chitosan (CHT) and alginate (ALG) have been widely employed as LbL ingredients to shape a wide repertoire of size- and shape-tunable electrostatic-driven multilayered assemblies by exploring their opposite charge nature. However, the insolubility of CHT in physiological conditions intrinsically limits the range of bioapplications of the as-developed CHT-based LbL structures. Herein, we report the preparation of free-standing (FS) multilayered membranes made of water-soluble quaternised CHT and ALG biopolymers for controlled release of model drug molecules. The influence of the film structure in the drug release rate is studied by assembling two distinct set-ups of FS membranes, having the model hydrophilic drug fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) either as an intrinsic building block or added as an outer layer after the LbL assembly process. Both FS membranes are characterised for their thickness, morphology, in vitro cytocompatibility, and release profile, with those having FITC-BSA as an intrinsic LbL ingredient denoting a more sustained release rate. This work opens up new avenues for the design and development of a wide array of CHT-based devices for biomedical applications, overcoming the limitations associated with the insolubility of native CHT under physiological conditions.