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A novel method has been developed to monitor the nucleic acids ligation process. Molecular beacon was employed here to convert the ligation information into fluorescence signal quickly and quantitatively. This method provides effective and original approach to researching the dynamic ligation process and the interactions between nucleic acids and ligase. An analytical method for T4 DNA ligase based on this way has been built up with a linear detection range from 2.3×10?4 U/mL to 0.23 U/mL. It is rapid and sensitive to detect 2.8×10?5 U T4 DNA ligase in 10 min.