<p>Recombinant enzymes and proteins are increasingly used in the key areas of biotechnology, environmental science, and biomedicine. To facilitate purification, recombinant proteins are often expressed with various affinity tags, with the His-tag being the most common. Melanin has emerged as a promising biomaterial with different excellent physicochemical properties. In this study, we successfully developed and characterized a melanin/alginate-based material incorporating cobalt ions (Co-M/A) as an alternative stationary phase for His-tagged protein purification. Co-M/A beads exhibited defined morphology and structural stability, which were critical for reproducible protein binding. EDX, XRF, XRD, and XRF analyses confirmed the presence of cobalt species in Co-M/A particles, while FTIR spectra suggested evidence of Co²⁺ coordination with carboxyl and hydroxyl groups in the M/A matrix. After loading the protein solution on the Co-M/A beads, the emergence of amide I and amide II peaks was observed, indicating the successful capture of His-tagged proteins through specific coordination between histidine residues and Co²⁺ ions. Functionally, Co-M/A demonstrated high specificity toward His-tagged proteins of different sizes and structures, while showing negligible non-specific binding to non-tagged proteins. Impressively, the purity of eluted proteins exceeded 95.3% after a single purification step using Co-M/A beads. Importantly, the functional activities of purified proteins, including nanobodies (A8) and enzymatic proteins (Lc-BoNT/A), were well preserved as confirmed by SDS-PAGE analysis and Western blotting analyses, ensuring downstream applications. In conclusion, this study demonstrates the successful development of Co-M/A beads as a promising stationary phase for His-tagged protein purification with high efficiency.</p>