Experimental setup for evaluating four-stage thermoelectric cooler’s performance was designed. Effects of input power, heat dissipation condition and heat load on the temperature difference (ΔT) of four-stage thermoelectric coolers’ hot and cold faces were obtained experimentally. The result shows that, the ΔT increases as the input power increases. A linear relationship exists between input current and feedback voltage. In different cooling conditions, the ΔT of thermoelectric cooler (TEC) increases with the temperature of hot face. As the temperature increasing on hot face is 1K, the ΔT increasing of TEC can be about 0.5K. Meanwhile, the power consumption of TEC also increases slightly. Water condensation can be prevented in either dry nitrogen environment or vacuum environment, but the vacuum level has great influence to the ΔT, especially in low operation temperature. The better the vacuum level is, the smaller the convection heat loss has. When the operation temperature of focal plane array (FPA) is lower than 220K, it is prior to use vacuum packaging. Considering the Joule-heat of readout circuit and the heat loss of wire conduction, the minimum working temperature of FPA can reach below 200 K when the temperature of the hot face is 285K. And the coefficient of performance (COP) of TEC can increase sharply from 0.8% to 4% when the controlled operation temperature is 220K rather than 200K.