BACKGROUND The quantification of gene expression is an important tool in the evaluation of the immune response to vaccines. Reliable reference genes for gene expression studies in mouse draining lymph nodes after vaccination have not been reported. RESULTS The utility of seven potential reference genes was investigated using commercially available Taq-man primer/probe mixes. Results were evaluated with RefFinder, a web-based program including multiple algorithm methods such as geNorm, NormFinder, BestKeeper and the comparative delta-Ct. Further assessment was done by applying the candidate reference genes in relative expression calculations with genes related to the magnitude and longevity of the humoral immune responses. The ubiquitin C gene, Ubc, was found to be the most reliable reference gene when validated with well-known genes that are expressed at relatively low levels after vaccination. The optimal time of sample collection varied depending on the function of the target genes. CONCLUSIONS This study identified Ubc as the most reliable reference gene and provides useful information for studies examining immunological gene expression in the draining lymph nodes after vaccination in mice.