Polyamines (PAs) play critical roles in plant growth, somatic embryogenesis (SE), etc. Previous studies have demonstrated that exogenous PAs could promote SE in plants. However, the effects of PAs on Ginkgo biloba L. SE are still unknown, especially in the switch from the initial callus (IC) to the embryogenic callus (EC) stage or to the globular embryo (GE) stage. This work identified 34 genes involved in PAs metabolism in G. biloba using genome-wide analyses. These genes were clustered into six families and found to be unevenly distributed across 11 of the 12 chromosomes on the plant. These families contain 539 cis-acting elements that mainly respond to phytohormones, abiotic stress, meristem expression, etc. RNAseq analysis revealed that the expression of GbADC2, GbSAMDC2, GbSPMS1, GbCuAO1 and 3, and GbPAO3, 8, 6 and 13 genes in G. biloba were higher in the GE stage than in the IC stage. In addition, 1.0 mg·L⁻¹ spermine (Spm3) could promote the conversion of IC to EC, while 0.01 mg·L⁻¹ putrescine (Put1) could facilitate the transition from IC to EC and then to GE. During the conversion of IC to EC or to GE, higher levels of abscisic acid (ABA), superoxide dismutase (SOD), and peroxidase (POD) and lower levels of indole-3-acetic acid (IAA), gibberellin (GA3), and zeatin (ZT) were observed; concurrently, the H₂O₂ level was also observed to be high. Gene expressions of GbSPMS2, GbCuAO3, and GbPAO6 and 8 were upregulated, while GbADC2 expression was downregulated in the EC or GE stages under Spm3- or Put1- treatment. These results illustrate that exogenous PAs might alter the levels of the endogenous polyamine pool and lead to H₂O₂ production, which caused a certain oxidative stress. However, SOD and POD balanced H₂O₂ production and maintained homeostasis. The PAs–H₂O₂–ABA module might coordinate the regulation of early in of G. biloba. These relations were discussed in this work. These findings provide a foundation for comprehending the roles of PAs gene families in the key nodes of early SE in G. biloba.