Transcription termination is a critical step for gene regulation and genome integrity among all kingdoms of life. In Saccharomyces cerevisiae, one of the major termination pathways is accomplished by Sen1 helicase, a homolog to human Senataxin (SETX), defection of which raises the diseases for the central nervus system of human. Although it has been proposed that Sen1 translocates along nucleic acids by consuming adenosine triphosphates (ATPs) during termination, the mechanism for this translocation activity of Sen1 has not been well understood. In this work, our aim is to investigate the mechanism of Sen1 translocation by measuring the interactions between Sen1 and different types of nucleic acids by polyacrylamide gel electrophoresis (PAGE) assay or single-molecule Fӧrster resonance energy transfer (FRET) assay. We firstly observe the unwinding activity of Sen1 on a tailed duplex DNA in the presence of 1 mM ATP via PAGE assay, where the translocation activity of Sen1 is involved. As the binding activity is crucial for translocation, then we examine the binding affinity of Sen1 to the single-stranded DNA via PAGE assay, revealing a stable binding of Sen1 with an occupied length of nucleic acids of less than 24 nt. In the presence of 1 µM ATP, we observe that Sen1 dynamically binds to and dissociates from the tailed duplex DNA in the single-molecule FRET assay. By titrating ATP concentrations from 1–500 µM, we observe a gradual decrease in the mean durations of Sen1 binding, suggesting an ATP-dependent binding affinity of Sen1 to single-stranded DNA. We then fit these mean durations to the classical Michaelis-Menten model and obtain a minimum binding duration of (0.18 ± 0.01) s at saturating ATP concentrations and K _mof (13.1 ± 0.1) µM for the ATP-dependent binding of Sen1. This result is consistent with that from a translocation activity of Sen1. Taking into account the translocation length of the half of the single-stranded tail, i.e. 13 nt, a mean rate of 70 nt/s is estimated. Reversing the translocation direction, we observe an increase in the duration of Sen1 binding to the single-stranded tail, which suggests an impediment of DNA duplex in front of Sen1 translocation or the possible duplex DNA unwinding activity of Sen1. Our quantitative measurements on Sen1 translocation are helpful in deepening our understanding of the mechanism of eukaryotic transcription termination by Sen1.