This paper aims to solve the problems of rising temperature irreversibly and easily affected by the external environment, which result from cold storage agent as the cold source in current low-temperature biological transfer boxes. An intelligent portable low-temperature biological transfer box based on a micro vapor compression refrigeration system was designed and built,, the cooling and cyclic test experiments were conducted, and the accuracy and reliability of the digital display temperature were improved through the polynomial fitting. When −20 °C is set, the first cooling time is 2.9 h, the single cycle time is 104 min, the temperature difference is about 3 °C between the inner wall of the cavity and the hollow environment. The internal environment temperature change is relatively stable. The designed transfer box can store biological materials for a long time, realize real-time monitoring and control of internal temperature and remote interactive functions during packaging and transportation.