The work aims to develop a multi-temperature insulation box and study its temperature control effects. The influences of the placement of coolant, the external environment and the mass ratio of coolant to cold fresh products on the temperature control effect of insulation box were studied by the single-factor experiment. The research results showed that, the combination of side placement and top placement was the best way to place the coolant with the best heat preservation effect. When the mass ratios of coolant to cold fresh product were 5∶6 (cold storage area) and 8∶5 (frozen area) in EPP multi-temperature insulation box with a thickness of 30 mm in the external environment (temperature: 23 ℃, relative humidity: 50%), the holding time reached 24 h or more. The holding time of each test point of the insulation box prolonged with the increase of the coolant mass, and the holding time of the cold storage area and the frozen area could reach as long as 44 h and 28 h, respectively. The mathematical model of external environment temperature-mass ratio of coolant to cold fresh product-holding time was established. The multi-temperature insulation box can meet the temperature control requirements of short-distance express transportation of cold fresh products.