[Objective] DNA phosphorothioation (PT) is a novel DNA modification in which a non-bridging oxygen atom on the phosphodiester bond is replaced with sulfur atom. PT modification serves as a constitute element of bacterial restriction-modification (R-M) defensive system, but more biological functions of PT modification are awaiting exploration. The methods currently used for the identification of PT-DNA are complicated, time-consuming and labor-intensive. Enzyme-linked immunosorbent assay (ELISA) has been known for its merits of easy handling, low cost and time-saving, which could be used for new PT-DNA identification method development. The homologous protein of PT-dependent REase ScoMcrA from Streptomyces coelicolor can be found in Streptomyces gancidicus, which suggested that this homologous protein presumably encodes the sulfur-binding domain (SBD) to specifically recognize sulfur atom on PT-DNA. In this study, the PT-DNA binding activity of SBD from S. gancidicus (Sg-SBD) was characterized qualitatively and quantitatively, respectively, which served as an assessment of its potential for the PT-DNA ELISA method development. [ Methods] Sequence alignment of ScoMcrA-SBD and Sg-SBD was conducted. The homology modeling was used to compare the possible structure of Sg-SBD with that of ScoMcrA-SBD. ScoMcrA-SBD and Sg-SBD were heterologously expressed in E. coli and purified. Using the purified Sg-SBD, the electrophoresis mobility shift assays (EMSA) was conducted firstly. Then, the biolayer interferometry (BLI) analysis was used to confirm the binding affinity of Sg-SBD for PT-DNA quantitatively. [Results] Bioinformatics analysis revealed that Sg-SBD contains conserved amino acid residues that bind to sulfur atoms, and the structure of Sg-SBD was similar with that of ScoMcrA-SBD. The EMSA showed that Sg-SBD can bind PT-DNA and form an obvious migration band, suggesting its PT-DNA binding activity. BLI data further confirmed that Sg-SBD possessed higher binding affinity to PT-DNA than that of ScoMcrA-SBD. Moreover, Sg-SBD exhibited no affinity to non-PT-DNA. [Conclusion] Sg-SBD could specifically bind PT-DNA, and the binding affinity could be comparable to that of antigen and antibody, which suggested the potent potential for ELISA method development for rapid detection of PT-DNA.