The paper aims to explore the influence of temperature field and effect of thermal insulation in mul-ti-temperature field insulation box under different conditions. The EPP incubator with the refrigerated area (0-8 ℃) and frozen area (below 0 ℃) in the double temperature zone insulation box was taken as the research object, and a geometric model was established by ANSYS FLUENT. The reliability of the model was verified by measuring the experimental holding time. Three factors, including different refrigerant placement, different box materials and different external environment temperature, were selected to carry out simulation calculation and analyze their influences on the internal temperature of the insulation box. Through simulation, it was concluded that the internal air temperature field was the most uniform and the insulation effect was the best when the refrigerant was placed around the sides. The insulation time was increased by 41.37% and 51.59% compared with the lowest edge refrigerating area. Among the three kinds of box materials, the insulation box made of EPU material had the best insulation effect, with the insulation time increased by 36.84% and 41.24% compared with the cold storage area of EPP material. With the increase of the external temperature from 23 ℃ to 40 ℃, the insulation effect of the insulation box in the refrigerated area decreased by 59.73%, and that in the frozen area decreased by 55.04%. The numerical simulation of the temperature field of the incubator in the multi-temperature region can be carried out by means of computer simulation. Its result can provide certain theoretical foundation for application of different temperature in the box of multiple temperature zones.