Taq pol, MMLV- RT and human pol β are polymerases lack of 3′-5′ exonuclease activity. The Taq polymerase binding with DNA template-primer (T-P) with 1, 2, or 3 mismatched base/bases at the terminus of primer was studied by surface plasmon resonance (SPR) biosensor, comparing with the binding with full matched DNA T-P. The experiment showed that the affinity of Taq pol binding with DNA T-P decreases when the number of mismatched bases increased, indicating that Taq pol preferred to binding with matched DNA T-P. With “incorrect” dNMP, the kinetics of Taq pol binding with matched DNA T-P could be analyzed by 1∶1 Langmuir model while the kinetics of MMLV RT－ binding with matched DNA T-P might involve conformation change. Conformation change model fitted well with Taq pol and MMLV－ RT-DNA binding curves in the presence of “correct”dNMP. The binding affinity was 20 times and 64 times higher than that without dNMP, respectively, indicating “correct” dNMP induced the conformation change of polymerse-DNA complex and enhanced the binding tightness. In the presence of dNMP, the kinetics of pol β binding with DNA T-P changed significantly and pol β grasped DNA closely.