In the present study, a technique of mid-infrared spectroscopy for detection of urea in milk was put forward. Eighteen adulterated milk samples with added different content of urea (1～18 g·L-1) were prepared. The mid-infrared spectra of these milk samples were measured. The infrared characteristics of pure milk and adulterated milk were studied, and analysis and comparisons of the differences were carried out. The absorption peak area (A1 562) of 1 562 cm-1, which was assigned to the C—N stretching vibration for urea, and the absorption peak area (A1 464) of 1 464 cm-1, which was assigned to the CO stretching vibration for amide Ⅲ’, were calculated by origin. Linear fitting of relation was made between A1 562/A1 464 and urea concentration in milk. The results show that the A1 562/A1 464 is linear with urea concentration in milk, with a regression coefficient of 0.96. The study is important to improving quality of dairy products and protecting the benefit of consumers, and takes on crucial social significance and application prospect.