Abstract: The present invention relates to a method for a non-specific amplification of a natural short-fragment nucleic acid, comprising the following steps: (1) performing end repair on the natural short-fragment nucleic acid to obtain an end-repaired nucleic acid; (2) connecting the end-repaired nucleic acid to a double-stranded linker to obtain a ligation product, in which each strand of the double-stranded linker contains only three bases; (3) performing PCR amplification on the ligation product using a PCR primer labeled with deoxyuridine to obtain a PCR product, in which the PCR primer is completely or partially complementary to a strand of the double-stranded linker and contains only three bases; and (4) digesting the PCR product by using an enzyme having a deoxyuridine cleavage function, followed by digesting the PCR product by using an enzyme with both 5??3? polymerase activity and 3??5? exonuclease activity in the presence of a deoxynucleotide solution to obtain a non-specific amplification product of the na