Resazurin is prepared by acid-catalyzed condensation between resorcinol and 4-nitrosoresorcinol followed by oxidation of the intermediate with manganese(IV) oxide:
Treatment of the crude reaction product with excess sodium carbonate yields the sodium salt of resazurin, which is typically the commercial form of the dye. Running the condensation step in alcohols is possible but results in lower yields of the product; in pure water or acetic acid, the reaction does not proceed satisfactorily.