Abstract: The experiments were carried out to study the effects of application of mixed water retention agent and pearlite on the emergence rate and the growths of flue-cured tobacco seedlings by the floating system and to provide a theoretical base for the new seedling medium. The results showed that: compared with peat medium, application of mixed water retention agent and 30% pearlite could had the same emergence rate and reduced the growth period by 0~2 d, thus having a higher or the same effects on agronomic character conditions and dry matter. The chlorophyll content of tobacco seedlings, the NR enzyme’s activity, and the SOD activity also increased by 0.24 mg/g, 1.73•?g/g FW•h and 31.3 ?g/g FW treated with application of mixed water retention agent and 30% pearlite, compared with the treatment of peat medium. Therefore, application of mixed water retention agent and pearlite could replace peat medium.