Ferredoxin is a typical iron-sulfur protein that is ubiquitous in biological redox systems. This study investigates the in vitro assembly of a [Fe₂S₂] cluster in the ferredoxin from Acidithiobacillus ferrooxidans in the presence of three scaffold proteins: IscA, IscS, and IscU. The spectra and MALDI-TOF MS results for the reconstituted ferredoxin confirm that the iron-sulfur cluster was correctly assembled in the protein. The inactivation of cysteine desulfurase by L-allylglycine completely blocked any [Fe₂S₂] cluster assembly in the ferredoxin in E. coli,confirming that cysteine desulfurase is an essential component for iron-sulfur cluster assembly. The present results also provide strong evidence that [Fe₂S₂] cluster assembly in ferredoxin follows the AUS pathway.