Single-leaf photosynthetic rate significantly affected by leaf location, leaf aging, and environment is insufficient to characterize the photosynthetic capacity and material accumulation efficiency of the entire plant. A plant population photosynthetic rate measurement system (PPPMS) was developed based on the carbon assimilation process using a transparent assimilation box. The PPPMS collected the data on light intensity, CO2 concentration, temperature and humidity in the assimilation box using a high-precision light radiation sensor and SCD30 to achieve the accurate control of the light environment, measurement of the carbon assimilation process, temperature and humidity control and so on. The synchronous experiment was carried out with the LI-6800 closed-circuit carbon assimilation measurement system, and the population photosynthetic rate was determined using linear fitting with the CO2 changes. The performances of the PPPMS and the detection accuracy of carbon assimilation process were evaluated at various lights, temperatures and crop types. The findings revealed that the system’s air tightness and light regulation accuracy were satisfactory, with the maximum air leakage rate of 0.0473μmol/(mol·min), the maximum single air leakage (6min) of 0.2838μmol/mol. And the average standard deviation of light regulation accuracy was 2.71μmol/(m2·s), which can meet the detection of plant carbon assimilation process. In the linear correlation analysis, the R2 of CO2 exchange capacity fitting of single and multiple lettuces were 0.988 and 0.874, respectively, with the root mean square error (RMSE) of 5.82μmol/mol and 5.80μmol/mol, while the fitting R2 of tomato was 0.952, and the RMSE was 3.39μmol/mol. The results showed that the system’s measurement performance was comparable to that of LI-6800 system, and the detection performance in upright plants was superior to that of leafy vegetables. Under varied temperatures and lighting, the average mean absolute errors (MAEs) of tomato and lettuce computed by light response curves between the PPPMS and LI-6800 system were 0.45μmol/(m2·s) and 0.35μmol/(m2·s),respectively, and the mean value of fitting R2 was not less than 0.95. It was demonstrated that the method can accurately and consistently measure the plant population’s light response curve.