Objective The system of Strep-Tactin and StrepII tag-SSB proteins binding (ST-SSB) was established to isolate the purified single-stranded DNA in a single step with low cost and high efficiency. Results We demonstrate that in the presence of large amounts of dsDNA, the ssDNA binding specificity of Escherichia coli (E. coli) single stranded DNA binding (EcSSB) protein was stronger than gene-5-protein (g5p). ST-SSB system relies on the affinity between Strep-Tactin, StrepII tag-SSB protein and ssDNA in binding buffer. Here, we successfully isolated the purified ssDNA from mixed DNA (ds- and ss-DNA form) samples and asymmetric polymerase chain reaction (aPCR) products. This system can purify ssDNA in a single tube within 1 h, and the recovery efficiency of purified ssDNA was around 60%. Conclusions The ST-SSB system has obvious advantages of high efficiency and one-step purification to recycle any ssDNA.