The work aims to study the effect of addition of CO2 generation agent on the ball quality and metabolic properties in the process of transporting broccoli and screen the proper concentration of CO2 in the package during such process. Broccoli was used as experimental material to study the effects of CO2 (carbon dioxide generation agent) at different concentrations on physiological characteristics and quality of broccoli ball under simulated transportation conditions after harvest. Keeping a low volume fraction (3% and 6%) of CO2 could better inhibit the respiratory intensity of broccoli during post-harvest transportation, delay the time and rate of increase of ethylene release, inhibit the degradation of chlorophyll in the flower ball, maintain the color of the flower ball, and reduce the decay index of broccoli during transportation. According to further research results, the treatment with CO2 (volume fraction: 3%) also delayed the increase rate of malondialdehyde (MDA) content in the flower ball during shelf life. A certain concentration of CO2 (3%) may improve the scavenging ability of active oxygen in broccoli tissue by inducing the catalase (CAT) and peroxidase (POD) activities, thus maintaining the quality of the flower ball. The higher concentration of CO2 (9% and 12%) had a better fresh-keeping effect in the first 3 days of transportation, but later promoted the metabolic rate (respiratory intensity and ethylene production) of the flower ball, accelerated the yellowing and decay of the flower ball and showed the signs of injury. The addition of CO2 (volume fraction: 3%) generation agent to the package before transportation can better reduce the metabolic level of broccoli during normal temperature transportation and maintain the quality of flower balls, thus prolonging the transportation time.