The invention discloses a method for preparing a dengue hemorrhagic fever virus II nucleic acid molecular characteristic standard sample. The dengue hemorrhagic fever virus II nucleic acid molecular characteristic standard sample is prepared according to the steps of synthesis of sequences, cloning of vectors, connection of target fragments and vectors, plasmid transformation, extraction and freezing dried storage of recombinant plasmids and the like. A standard sample that contains virus characteristic sequence information and is suitably used for analyzing the virus and the content thereof in a sample is prepared according to the method; the standard sample provided by the invention has good homogeneity, high stability and good purity, can be stored for long term, and is prepared according to the simple preparation method; a good standard sample is provided for detection research, medical research and the like of dengue hemorrhagic fever virus II type so as to achieve comparison of results from different laboratories and guarantee the quality control of laboratories; and a standard sample is also provided for fast and accurate detection of dengue hemorrhagic fever virus II type for inspection and quarantine institutions, import and export trade and the like.