Recombinant proteins are crucial in biomedical research, highlighting the importance of efficient expression and purification methods. Here, we present a protocol for the expression of recombinant macrophage migration inhibitory factor (MIF), followed by its purification. Our optimized approach incorporates three chromatographic purification steps: anion and then cation exchange chromatography followed by size exclusion chromatography (SEC). Protein validation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent immunoblotting confirms the production of pure MIF protein.