Peony (Paeonia lactiflora Pall.) seeds stored for one or two years and newly harvested seeds were used as experiment materials. The seeds were treated with low temperature (-20 ℃ or -40 ℃) and then stored in the sand for elucidating the methods for promoting rooting process. Physiological and biochemical indexes were analyzed during different rooting stages for investigating the internal mechanisms. The results showed: (1) Oneway analysis of variance showed storage years, treatment temperature and treatment time length performed obviously significant main effects together or independently on rooting index, rooting rate and firstseed rooting time of peony seeds, and storage years performed strongest influence. (2) For peony seeds stored in the same year, treatment of -40 ℃ treatment for 30 days before sand storage performed the best rooting efficiency with the highest rooting rates of 88.33%. When under the same treatments, the rooting efficiency of oneyearstorage peony seeds is better than those of other storage time length. (3) Physiological and biochemical analysis showed that during the seed rooting process, for the peony seeds stored for the same time length, the water content kept raising, the soluble sugar content increased first and then decreased, the starch content kept decreasing, the soluble protein content decreased first and then increased; peroxidase (POD) activity increased first and then decreased, catalase (CAT) activity decreased after an increase. Conclusion: the treatment of -40 ℃ for 30 days can effectively break the dormancy of Paeonia lactiflora Pall. seeds and promote the rooting process, and oneyearstorage seeds performed the best rooting efficiency.