The invention discloses a method for detecting discontinuous multiple DNA loci by combining an overlapping extended PCR with Sanger sequencing. The method includes a pair of primers for full-length amplification of the overlapping extended PCR, and the nucleotide sequence is shown as SEQ ID No.1-2. The discontinuous loci are connected into a DNA fragment through one-time PCR, and the fragment where the multiple detection loci are located is subjected to one-time Sanger sequencing analysis. By means of the method, any 2-4 fragments containing the detection loci can be spliced together to form fusion fragments, rapidness and simpleness are achieved, restriction endonuclease is not needed, no non-target gene DNA sequence exists, thus the detection breaks through the restriction of the Sangersequencing reading length, and multiple discontinuous DNA loci can be simultaneously detected through one Sanger sequencing reaction.