OCT4A has been known to play a critical role in the maintenance of pluripotency of embryonic stem cells. Recent research has shown that OCT4A is also expressed in partial tumor cell lines and tissues. This study is aimed to develop a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for relative quantitative detection of OCT4A mRNA and discrimination from OCT4B, pseudogene, and genomic contaminations. A locked nucleic acid (LNA)-modified probe was designed to discern the single base difference 352A/C to identify OCT4A mRNA. An exon-junction primer was designed to avoid false positive caused by genomic contaminations. In addition, a house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured in parallel to normalize the differences between samples and operations. Experiments showed that the newly established RT-PCR assay amplified the OCT4A mRNA selectively; OCT4A analogues gave negative signals. Cell lines nTERA-2 and HepG2 showed positive results in OCT4A expression, while for HeLa and 293 cell lines, as well as primary peripheral blood mononuclear cells (PBMCs), OCT4A expression was negative. Additionally, the relative quantity of OCT4A mRNA was calculated by cycle threshold (Ct) method and house keeping gene normalization. This technique proved to be effective for relative quantitation of OCT4A mRNA with high specificity.