Proteins and amino acids are essential to humans and all living beings. Moreover, these compounds are present in the pharmaceutical, food, and cosmetic industries. Proteins need to be purified for use in industry. This work aims to synthesize and apply a nano-biocomposite based on graphene oxide (GO) and agar (polysaccharide) for protein separation and purification. The hydrogel composite was tested for myoglobin (Mb) adsorption, the effect of changing the initial pH in Mb adsorption, and the ionic strength was evaluated. The adsorptive capacity values were higher at pH=5 and 6 and decreased at higher pH values. The increase in NaCl concentration did not affect the adsorptive capacity significantly. The composite exhibited a notorious value of maximum experimental adsorption capacity in the equilibrium of over 790 mg g−1 at pH=5 and 25 °C. As GO interacts with the polymeric agar matrix, resulting in a 3D material, the material developed could adsorb Mb in a fixed bed column and single and binary (BSA+Mb) systems. There are indications that the GO-based composite possibly forms a complex with the peptide ligands of myoglobin, forming a protein corona, making Mb desorption difficult.