For developing a canopy photosynthesis model, an efficient method to measure the photosynthetic rate in a growth chamber is required. The objective of this study was to develop a method for establishing canopy photosynthetic rate curves of romaine lettuce (Lactuca sativa L.) with light intensity and CO 2 concentration variables using controlled growth chamber. The plants were grown in plant factory modules, and the canopy photosynthesis rates were measured in sealed growth chambers made of acrylic (1.0 x 0.8 x 0.5 m). First, the canopy photosynthetic rates of the plants were measured, and then the time constants were compared between two application methods: 1) changing light intensity (340, 270, 200, and 130 μmol•m -2 •s -1 ) at a fixed CO 2 concentration (1,000 μmol•mol -1 ) and 2) changing CO 2 concentration (600, 1,000, 1,400, and 1,800 μmol•mol -1 ) at a fixed light intensity (200 μmol•m -2 •s -1 ). Second, the canopy photosynthetic rates were measured by changing the light intensity at a CO 2 concentration of 1,000 μmol•mol -1 and compared with those measured by changing the CO 2 concentration at a light intensity of 200 μmol•m -2 •s -1 . The time constant when changing the CO 2 concentration at the fixed light intensity was 3.2 times longer, and the deviation in photosynthetic rate was larger than when changing the light intensity. The canopy photosynthetic rate was obtained stably with a time lag of one min when changing the light intensity, while a time lag of six min or longer was required when changing the CO 2 concentration. Therefore, changing the light intensity at a fixed CO 2 concentration is more appropriate for short-term measurement of canopy photosynthesis using a growth chamber.