The measurement of net ecosystem exchange (NEE) of field maize at a plot-sized scale is of great significance for assessing carbon emissions. Chamber methods remain the sole approach for measuring NEE at a plot-sized scale. However, traditional chamber methods are disadvantaged by their high labor intensity, significant resultant changes in microclimate, and significant impact on the physiology of crops. Therefore, an automated portable chamber with an air humidity control system to determinate the nighttime variation of NEE in field maize was developed. The chamber system can automatically open and close the chamber, and regularly collect gas in the chamber for laboratory analysis. Furthermore, a humidity control system was created to control the air humidity of the chamber. Chamber performance test results show that the maximum difference between the temperature and humidity outside and inside the chamber was 0.457 °C and 5.6%, respectively, during the NEE measuring period. Inside the chamber, the leaf temperature fluctuation range and the maximum relative change of the maize leaf respiration rate were −0.3 to 0.3 °C and 23.2015%, respectively. We verified a series of measurements of NEE using the dynamic and static closed chamber methods. The results show a good common point between the two measurement methods (N = 10, R2 = 0.986; and mean difference: ΔCO2 = 0.079 μmol m−2s−1). This automated chamber was found to be useful for reducing the labor requirement and improving the time resolution of NEE monitoring. In the future, the relationship between the humidity control system and chamber volume can be studied to control the microclimate change more accurately.