RAW264.7 is a macrophage strain derived from mice tumour and shows a significant ability in antigen uptake. Real-time quantitative PCR (RT-qPCR) is one of the most commonly used methods in gene studies and requires suitable reference genes to normalize and quantitate the expression of gene of interest with sensitivity and specificity. However, suitable reference genes in RAW264.7 cells have not yet been identified for accurate gene expression quantification. In the current study, we evaluated expression levels of ten candidate reference genes in RAW264.7 cells under different conditions. RT-qPCR results indicated significant differences in the expression levels among the ten reference genes. Statistical analyses were carried out using geNorm, NormFinder, and BestKeeper software to further investigate the stability of the reference genes. Integrating the results from the three analytical methods, cytochrome c-1 and hydroxymethylbilane synthase were found to be the most stable and therefore more suitable reference genes, while ribosomal protein L4 and cyclophilin A were the least stable. This study emphasises the importance of identifying and selecting the most stable reference genes for normalization and provides a basis for future gene expression studies using RAW264.7 cells.