Metals play a vital role in many industries, but their extraction through traditional mining methods poses significant environmental challenges. To address these issues, alternative sources such as coal fly ash (CFA) need to be explored for the recovery of transition metals. In this study, we describe a novel green strategy for the selective separation and recovery of valuable metal ions from complex solutions using semi-synthetic proteins. Myoglobin (Mb), a small protein available from renewable sources, was conjugated to the high-affinity metal chelator SG-20 to create the semi-synthetic protein Mb-SG-20. Mb-SG-20 was characterized by gel electrophoresis, mass spectrometry, and proteomic analysis and found to contain a distribution of 4–7 equiv of SG-20 conjugated to Mb surface lysine residues. Binding and recovery properties of Mb-SG-20 for nickel (Ni) and copper (Cu) ions were characterized. The results showed that Mb-SG-20 captures super-stoichiometric amounts of Ni or Cu while maintaining a strong affinity for Ni or Cu ions. Furthermore, we demonstrate the recyclability of Mb-SG-20 as a Ni and Cu ion capture agent and provide proof-of-concept experiments using CFA leachate solution. This study presents a promising approach for the sustainable recovery of valuable metals from non-traditional sources, such as CFA.