Abstract: Polyamines such as putrescine, spermine, and spermidine play important roles in plant growth, development, and stress responses. To investigate the roles of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), the key enzymes involved in putrescine biosynthesis, in response to abiotic stresses such as high temperature, this study conducted the bioinformatic, transcriptional and protein level analyses of the GlADC and GlODC genes, as well as putrescine content detection in the seaweed Gracilariopsis lemaneiformis. One GlADC and one GlODC gene were identified in the G. lemaneiformis genome, which belong to the type III pyridoxal-dependent arginine decarboxylase family and the type III pyridoxal-dependent ornithine decarboxylase family, respectively. Quantitative real-time PCR analysis showed that both high temperature and high light stresses mainly upregulated the expressions of GlADC and GlODC, whereas nitrogen starvation downregulated their transcript levels. After prokaryotic expression, recombinant protein expression and purification, and antibody preparation, western blot showed that high temperature promoted the GlADC and GlODC protein levels, high light displayed no significant effect, yet low nitrogen downregulated or had no significant effect on the expression levels of the two proteins. Moreover, putrescine content increased under high temperature and high light stresses. These findings suggest that putrescine and its metabolic enzymes participate in the response of G. lemaneiformis to high temperature, high light, and low nitrogen conditions. This study will provide valuable insights into the metabolic pathways of putrescine and its roles in alga stress physiology.