RecQ5β is an essential DNA helicase in humans,playing important roles in DNA replication,repair,recombination and transcription.The unwinding activity and substrate specificity of RecQ5β is still elusive.Here,we used stopped-flow kinetic method to measure the unwinding and dissociation kinetics of RecQ5β with several kinds of DNA substrates,and found that RecQ5β could well unwind ss/dsDNA,forked DNA and Holiday junction,but was compromised in unwinding blunt DNA and G-quadruplex.Rec5β has the preferred unwinding specificity for certain DNA substrates containing the junction point,which may improve the binding affinity and unwinding activity of RecQ5β.Moreover,from a comparison with the truncated RecQ5β1-467,we discovered that the C-terminal domain might strongly influence the unwinding activity and binding affinity of RecQ5β.These results may shed light on the physiological functions and working mechanisms of RecQ5β helicase.