Abstract: Gracilariopsis lemaneiformis is the second most cultivated seaweed in China. In this study, the role of polyamines (PAs) in promoting carpospore release in G. lemaneiformis was investigated. The results showed that 300 μmol/L PAs (putrescine:spermine:spermidine 1:1:1) effectively increased carpospore release in G. lemaneiformis. Exogenous PAs influenced the photosynthetic system of G. lemaneiformis, with the photosynthetic capacity decreasing on the third day but largely recovering on the seventh day. Moreover, the contents of phycoerythrin (PE) and chlorophyll a (Chl a) significantly increased after three days. Additionally, the level of reactive oxygen species (ROS) significantly increased after PAs stimulation, and the activities of antioxidant enzymes and levels of antioxidant substances, such as superoxide dismutase (SOD), peroxidase (POD), malate dehydrogenase (MDH), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA), also changed accordingly. The increase of auxin, cytokinin, and salicylic acid contents, and the decrease in gibberellin content indicate that PAs may promote carpospore release by regulating phytohormones. Transcriptome analysis revealed that after three days’ PAs treatment, the expression of photosynthesis- and antioxidant-related differentially expressed genes (DEGs) was increased. In addition, the MAPK signaling pathway and other pathways initiated by Ca²⁺ were activated, regulating the levels of ROS in G. lemaneiformis. The DEGs related to cell wall degradation and synthesis also indicated that PAs can regulate carpospore release. These findings confirm the role of PAs in promoting carpospore release, reveal the associated physiological changes and potentially involved signaling pathways, and provide new insights for further research and commercial application on G. lemaneiformis reproduction.