A method of acquiring an unknown flanking sequence of a known sequence is disclosed. The method includes 1) designing two specific primers according to the known sequence which are labeled as SP1 and SP2, with the SP2 carrying a restriction enzyme recognition site, and designing a pair of reverse PCR primers which are F1 and D1 between the SP2 and a border sequence, 2) amplifying to obtain a PCR product 1 by adopting genome DNA as a template and by using any one degenerate primer and the specific SP1 primer, 3) amplifying to obtain a PCR product 2 by adopting the PCR product 1 as a template and by using the specific SP2 primer and a degenerate primer, with the degenerate primer in the step 3) being same with the degenerate primer in the step 2), but comprising a restriction enzyme recognition site, and 4) subjecting the PCR product 2 to digestion, adding T4 ligase, cyclizing, and amplifying by adopting the cyclized product as a template and by using the F1 primer and the D1 primer to obtain a PCR product 3, thus acquiring the flanking sequence. The method is advantaged by simple and easy operation, a low cost, a high success rate, short time, and the like.