In order to verify the effect of rootstock types on polyamine metabolism and floral bud dormancy of sweet cherry, this study collected the floral buds of sweet cherry variety ‘Royal Lee’ grafted in the dwarf rootstock Gisela 6 (G6) and the vigorous rootstock Mazzard as materials. Field investigations found that the dormancy process and florescence of the same sweet cherry variety grafted in dwarf and vigorous rootstocks are significantly different, and then we used the bioinformatics, gene cloning technique, realtime fluorescence quantification, subcellular localization methods to clone and do functional analysis of the key genes ［polyamine oxidase gene (PavPAOs) and arginine decarboxylase gene (PavADC)］ in the polyamine metabolism in sweet cherry floral buds. With online websites and bimolecular fluorescence complementation we predicted and verified proteins that may interact with PavADC. In addition, the content of free polyamines in floral buds was determined by high performance liquid chromatography. Results showed that: (1) the dormancy period of ‘MazzardRoyal Lee’ (75 d) is longer than that of ‘G6Royal Lee’ (69 d). And the flowering period of ‘MazzardRoyal Lee’ is later than ‘G6Royal Lee’. (2) The content of polyamines in floral bud of ‘G6Royal Lee’ is higher than that of ‘MazzardRoyal Lee’. In floral buds of ‘G6Royal Lee’, Put content was 1.2%-163.4% higher than that of ‘MazzardRoyal Lee’ and Spd content was 8.8%-261.1% higher than that of ‘MazzardRoyal Lee’. Spm was not detected in the floral buds of ‘MazzardRoyal Lee’, while was synthesized in ‘G6Royal Lee’ during January and March. (3) The key genes of sweet cherry polyamine metabolism PavPAO2, PavPAO4, PavPAO5 and PavADC were successfully cloned, and their lengths were 1 485, 1 611, 1 704, and 2 307 bp, respectively. Phylogenetic analysis showed that sweet cherry PavPAOs and PavADC are closely related to species such as Prunus persica, Prunus dulcis, and Malus domestica; subcellular localization analysis shows that PavPAO2 and PavPAO4 are located in the cell membrane, while PavPAO5 and PavADC are located in the nucleus. (4) qRTPCR analysis shows that the expression of PavPAO5 in floral buds of ‘G6Royal Lee’ is lower than that of ‘MazzardRoyal Lee’, while the expression of PavADC is higher than that in ‘MazzardRoyal Lee’ during dormancy. Throughout the dormant period, the overall expression of PavPAO2 and PavPAO4 were much lower than PavPAO5. It is speculated that PavPAO5 may be the dominant family member during the dormant period. (5) BiFC shows that PavADC could interact with PavSAMDC during the polyamine metabolism and we speculated that the two are involved in regulating the levels of polyamines in floral buds. Studies have shown that the types of sweet cherry rootstocks can affect the process of dormancy and flowering by affecting the process of polyamine metabolism. The high expression of key genes PavPAOs and PavADC can affect the level of polyamines in floral buds. PavADC can increase the content of polyamines to promote the release of dormancy and early flowering. The expression of key genes and changes in polyamine content during the process of polyamine metabolism can affect dormancy and florescence, and the increase in polyamine content can promote the release of dormancy and early flowering.