Abstract: Provided are a helicase mutant, a preparation method therefor, and use thereof in high-throughput sequencing. The helicase mutant has at least one cysteine on a surface of a three-dimensional structure of an amino acid sequence as set forth in SEQ ID NO: 1, the at least one cysteine being substituted with alanine. The cysteine is C133, C164, C292, C323, and/or C347. Preferably, the amino acid sequence of the helicase mutant is as set forth in SEQ ID NO: 6. The helicase mutant exhibits excellent DNA unwinding activity, good protein uniformity, reduced cross-linking interactions between protein molecules, enhanced adaptability with perforin, improved sequencing stability, as well as integrity and continuity of electrical signals. Consequently, it achieves a better sequencing performance. It can be utilized for the control and characterization of nucleic acids and applied in single-molecule nanopore sequencing.