The invention discloses a three-level flow sequence comparison method based on a many-core co-processor. The purpose of increasing the comparison speed of sequence comparison software is achieved. According to the technical scheme, sequence comparison is performed in a multi-threading manner by an MIC (microwave integrated circuit) many-core co-processor; three serial steps of reading sequences from a main memory to an MIC, comparing the sequences and writing a comparison result to the main memory in a sequence comparison process of the MIC are in a three-level flow mode, namely, sequences required for next comparison are read during sequence comparison, a previous comparison result is written into the main memory, and a reading and writing operation and a comparison operation are carried out simultaneously. By the three-level flow sequence comparison method based on the many-core co-processor, three main processes of sequence reading, sequence comparison and comparison result returning are carried out simultaneously, the comparison efficiency is improved, and the comparison time is shortened. Compared with a two-channel eight-core CPU (central processing unit), the three-level flow sequence comparison method has the advantages that the speed of a comparison process can be increased 2.3 times at least, a large amount of memory space is prevented from being copied, and the space-time efficiency of a procedure is improved.