An enzyme with strong single-stranded DNA (ssDNA) ligation activity would be advantageous for many molecular biology applications. However, currently available enzymes exhibit only limited activity. Here, we identified an enzyme with strong ssDNA ligation activity upon searching the databases for proteins homologous to TS2126 RNA ligase, the known enzyme with the highest yet limited ssDNA ligation activity. A ligase from Thermus phage phiLo (GenBank: AYJ73970) or Tph ssDNA ligase (SDL) depicted higher ssDNA ligation activity than TS2126 RNA ligase. This study suggests that SDL could be useful for various applications including sequencing for library preparation.