The invention discloses a multi-sample and multi-segment DNA methylation high-throughput sequencing method. The method can analyze DNA methylation states of multiple samples and multiple target segments at the same time in a library; the length of the segments can be about 500 bp, and the PCR recombination rate is low. The method further has the advantages of being small in initial sample capacity, high in pertinence, low in cost, capable of utilizing sequencing throughput fully and the like.