The invention discloses a method for determining an unknown sequence of the 5'-end flanking region on the basis of the known DNA sequence, wherein the method relates to the technologies such as restriction enzyme cutting, pUCm-T vector and nested polymerase chain reaction (PCR) amplification. The method comprises the following steps: a restriction enzyme is selected to perform enzyme cutting to the genomic DNA, Taq enzyme or Ex Taq enzyme are used to fill the purified enzyme cutting product and perform 3'-end A-adding reaction, the product is connected with the pUCm-T vector; a primer design software is utilized to select a sequence fragment in the upstream region of the T/A cloning site of the T-vector as 5'-end primer, two complementary sequences of the known sequence near the unknown sequence end are selected as 3'-end primer; and the pUCm-T vector connector is used as template, the designed primer is used to perform nested PCR amplification, and the amplified fragment is cloned and sequenced. The method is characterized by simple operations, wide application range, fewer operational limits, simple verification, unlimited unknown sequence, low cost and the like.