Micro-environment such as temperature influenced the growth and quality of plantlets in vitro. Chlorophyll fluorescence (ChlF) parameter is an important one for photosynthesis capacity in plant. The modulated chlorophyll fluorescence imaging system can be used for obtaining ChlF imaging and signal of plantlet in vitro because the container has light permeability. Therefore, the biological activity and growth condition of plantlet in vitro can be estimated by sterilely measuring the characteristics of chlorophyll fluorescence. This study determined the parameter of chlorophyll fluorescence in Orychophragmus violaceus plantlets in vitro preserved under different temperature levels (0, 4, and 8°C). The results showed that photosynthesis capacity in Orychophragmus violaceus plantlets in vitro preserved under 4°C condition were higher than that under 0°C or 8°C conditions. The plantlets in vitro preserved under 4°C condition maintained a high vitality to be subcultured.