Selection of a reliable reference gene for the normalization of RT-qPCR data is important to obtain accurate results in the study of gene expression. To elucidate the effects of temperature on the gene expression in Gracilaria lemaneiformis, this study aimed to find an appropriate reference gene from ten candidate genes, including actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 (EF1), eukaryotic translation initiation factor (eIF), histone (H2B), 18S ribosomal rRNA (18S rRNA), internal transcribed spacer 2 (ITS2), cytochrome c oxidase (COX), cytochrome b-5 reductase (CR) and phycoerythrin (PEB) of G. lemaneiformis under temperature stress. Programs geNorm and NormFinder were used to analyze the data of RT-qPCR. The results showed that the best reference gene combination was eIF, ACT for 32°C different treatments, GAPDH, ITS2, CR, and 18SrRNA for 8 °C different treatments, GAPDH, EF1 and ACT for different temperature treatments, and GAPDH and ACT for different life generations. This study provides the useful reference genes for future gene expression studies of G. lemaneiformis under temperature stress.