In order to improve the environmental conditions of tissue culture in vitro and to investigate the function of CO₂ in the culture, a CO₂ enrichment and real time control system was developed. Using this system, the growth experiment of subcultured grape (cabernet sauvignon) plantlets was carried out as compared with the traditional tissue culture method (non CO₂ enrichment). The results indicated that the system worked well, the required ranges of CO₂ concentration [(800～1 200) μL/L] could be obtained; and the CO₂ enrichment environment could promote the growth and photoautotrophic capability of the plantlets in vitro.