Exonuclease III (Exo III) has been generally used as a double-stranded DNA (dsDNA)-specific exonuclease that does not degrade single-stranded DNA (ssDNA). Here, we demonstrate that Exo III at concentrations above 0.1 unit/μL can efficiently digest linear ssDNA. Moreover, the dsDNA specificity of Exo III is the foundation of many DNA target recycling amplification (TRA) assays. We demonstrate that with 0.3 and 0.5 unit/μL Exo III, the degradation of an ssDNA probe, free or fixed on a solid surface, was not discernibly different, regardless of the presence or absence of target ssDNA, indicating that Exo III concentration is critical in TRA assays. The study has expanded the Exo III substrate scope from dsDNA to both dsDNA and ssDNA, which will reshape its experimental applications.