Abstract: In order to understand the prevalence and distribution of paratuberculosis in a large-scale dairy farm in Northern Xinjiang Uygur Autonomous Region, according to the principle of stratified sampling, the experiment randomly collected 258 serum samples of dairy cows of different ages. ELISA was used to detect serum antibody of paratuberculosis. Rectal scrapings were collected from 5 long-term diarrhea and lean cows. PCR method was applied to amplify 16 S rRNA gene, IS900 gene and subtype gene of Mycobacterium avium subsp. Paratuberculosis, and 16 S rRNA gene sequencing and homology comparison were carried out. The results showed that the positive rate of serum antibody in the farm was 17.44%（45/258）. The positive rate of paratuberculosis serum antibody in calves aged ＞3-6 months（38.24%, 13/34） was very significantly higher than those of adult cattle aged ＞1-2（7.89%, 3/38）, ＞2-3（9.72%, 7/72）, and ＞5-6 years old（4.55%, 1/22,P＜0.01）. The positive rate of paratuberculosis serum antibody in the calf stage was 31.48%（17/54）, which was significantly higher than those in the rearing stage（8.57%, 3/35） and late lactation stage（11.43%, 4/35,P＜0.05）. The positive rate of paratuberculosis serum antibody of first-born cattle（7.69%,5/65） was significantly lower than those of 0-born cattle（22.47%, 20/89） and second-born cattle（21.95%, 9/41, P＜0.05）. The positive rate of paratuberculosis serum antibody in the calf stage was significantly higher than that in the rearing stage（8.57%,3/35）, pregnancy stage（15.87%, 10/63） and lactation stage（14.15%,15/106,P＜0.05）;there was no significant difference between the rearing stage, pregnancy stage and lactation stage（P＞0.05）. The PCR test results of 5 rectal scrapes showed that 3 samples were bovine type Mycobacterium avium subsp. Paratuberculosis. The results suggested that the infection of paratuberculosis in this large-scale cattle farm was not optimistic, and it accured in cows of in different ages, production stages, parity and physiological status. The infection strain was bovine type Mycobacterium avium subsp. Paratuberculosis, which inferred that calf infection was an important risk factor affecting paratuberculosis infection in dairy cows.