The invention belongs to the technical field of biology and relates to a heavy chain single-domain antibody of a type-II dengue fever virus NS1 protein as well as a preparation method and application of the heavy chain single-domain antibody. The preparation method comprises the following steps of: biologically screening the recombination expressed dengue fever virus NS1 protein for many times by using an alpacas non-immunity heavy chain single-domain antibody library; picking by using an ELISA (Enzyme Linked Immunosorbent Assay) to obtain a specific bonded phage monoclone, and determining a DNA (Deoxyribose Nucleic Acid) sequence; and then, converting the DNA sequence into escherichia coli to express to obtain the heavy chain single-domain antibody. The heavy chain single-domain antibody obtained through screening can be efficiently expressed in the escherichia coli, can be used for preparing a colloid gold detection kit for detecting type-II dengue fever virus infection, and is higher in detection accuracy.