The cell adhesion molecules CD44v6 and integrin-beta 1 are associated with the progression and metastasis of cancer. A novel triplex real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify CD44v6 and integrin-beta 1 gene expression in peripheral blood mononuclear cells from 30 pancreatic cancer (PC) patients and 12 healthy individuals. The standard curve of the triplex qRT-PCR was constructed by optimizing the reaction condition and the amplification efficiency was 102.5, 101.1, and 100.6 % for CD44v6, integrin-beta 1 and endogenous gene (beta-actin) amplification. Nonspecific bands were not observed from the triplex qRT-PCR amplification and the detection limit of this assay was 100 copies. Expression levels of CD44v6 and integrin-beta 1 gene were significantly lower in healthy individuals than PC patients (P < 0.05). CD44v6 and integrin-beta 1 gene expression were not associated with the sex, age, and tumor position in PC (P > 0.05). CD44v6 gene expression was significantly associated with clinical stage, liver metastasis, and tumor size (P < 0.05). Integrin-beta 1 gene expression was significantly associated with clinical stage and liver metastasis (P < 0.05). This triplex qRT-PCR assay may provide a useful tool for diagnosis, prognosis, and therapeutic evaluation in PC.