In order to optimize the refrigeration efficiency and reduce the temperature difference between the cold and hot ends of the low temperature thermoelectric refrigeration test box, the effects of current, voltage and temperature difference on the refrigeration performance were calculated theoretically under the hot end temperature at 56.18 ℃ and 27.31 ℃. Three-dimensional mathematical heat transfer model was set up, and the effect of three kinds of thermoelectric cooler layout on the temperature distribution in refrigeration box was simulated. The results show that when the hot end temperature declines from 56.18 ℃ to 27.31 ℃, the refrigerating capacity decreases from 150.2 W to 139.5 W, the refrigerating coefficient decreases from 4.41 to 3.09, and the condensing temperature decreases from ?22.30℃ to ?41.90℃. In addition, the theoretical results prove that the reduction of the hot end temperature of thermoelectric cooler can produce lower condensing temperature, and the higher hot end temperature has better refrigeration performance.