Abstract: A primer for detecting fecal pollution in water, kit and high-throughput tracing method is disclosed, and belongs to the field of water pollution detection. The method mainly comprises the following steps: extracting DNA from water samples to be tested; using two pairs of universal primers for amplifying mitochondrial DNA to perform nested PCR amplification on the water sample DNA; performing high-throughput sequencing of the amplified products; performing annotation and alignment between sequencing data and a mitochondrial DNA database, and determining the sources of fecal pollution in water samples based on the alignment results. According to the present invention, nested PCR technology is utilized to amplify mitochondrial DNA with high sensitivity.