Abstract: In this paper, the rule of CO₂ concentrations and the effects of CO₂ enrichment concentration on the growth of sugar-free culture plantlets in the culture box with real-time control system after transplanting petunia, chrysanthmum and tomato plantlet were investigated, which was developed by Key Lab of Agricultural Bio-enviroment Engineering, Ministry of Agricultue, China Agricultural University. The day after transplanting, the CO₂ concentration started to decrease, speeding up the rate of decline in the second day and it dropped below 100 μL/L. In the first 4～5 days after transplanting, CO₂ conpensation dropped to between 30～40 μL/L. So CO₂ should be enriched on the second day after transplanting, otherwise, it would affect plantlet growth. In different CO₂ enrichment tests, tissue-culture is the best at CO₂ concentration of (650±50)μmol/mol under 80 μmol/(m²·s) Photosynthetic Photon Flux Density(PPFD).