D-dopachrome tautomerase (D-DT) is a multifunctional protein with various physiological roles, including immune system regulation and protection from oxidative stress. However, D-DT is also implicated in the progression of inflammatory conditions, cancers, and neurodegenerative diseases, where oxidative stress contributes to neuronal damage. Despite its importance, D-DT’s role in cellular processes and disease remains to be fully elucidated. For both basic research and exploring D-DT as a potential therapeutic target, access to recombinant, purified D-DT is invaluable. We present a detailed methodology for the bacterial expression of D-DT and its purification using ion exchange and size exclusion chromatography. Our systematic approach yields 8 mg of purified protein per liter of bacterial culture.