Transcriptional sequencing reaction for mass spectrometry analysis was compared using wild type and Y639F, F644Y, Y639F/F644Y mutant T7 RNA polymerases. Because 3´-dGTP, 3´-dUTP and 3’-dCTP except 3´-dATP were not commercially available, transcriptional sequencing with 300uM of GTP, ATP, UTP and 2´-dCTP as elongating nucleotide and 2mM 3’-dATP, 1mM ddGTP, 2mM ddATP, 0.5mM ddTTP and 0.2mM ddCTP as terminating nucleotides on linear and circular DNA template was performed using mutant T7 RNA polymerases. Transcription activity were 260.04 unit/㎍ for wild type, 194.12 unit/㎍ for Y639F, 193.8 unit/㎍ for F644Y, and 44.36 unit /㎍ for double (Y639F/F644Y) mutant T7 RNA polymerase. The order of sequencing reaction with 3´-dATP was Double (Y639F/F644Y) ＞ Y639F ＞ F644Y ＞ WT between 20 to 50 mer for circular DNA template and Double ＞ F644Y ＞ WT ＞ Y639F between 20 to 50 mer for linear DNA template. Double mutant had better transcriptional sequencing activity with 3´-dATP than wild and Y639F, F644Y mutants. Wild type and F644Y, Double mutant had non-specific termination but Y639F mutant can incorporate ddNTPs on circular DNA template. Y639F mutant can use ddNTPs on linear DNA template but wild type and F644Y, double mutant can not incorporate ddNTPs. Y639F had good transcriptional sequencing ability with 2´-dCTP as elongating nucleotide for better mass resolution between U and C and with ddNTPs instead of 3’-dNTPs as terminating nucleotides. This strategy will help for the development of mutant T7 RNA polymerases with high specificity and transcription activity for mass spectrometry analysis.