We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (T(m)) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the T(m) of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the T(m) difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.