Circulating tumor cells in the blood play a central role in the metastatic process. There have been no reports describing the relationship between lung metastasis and circulating osteosarcoma cells. We developed a system with a polymerase chain reaction assay based on an enzyme-linked immunosorbent assay (PCR-ELISA) to detect circulating osteosarcoma cells in a mouse metastatic model. Osf2/Cbfa1, hereafter called Osf2, a member of the runt family of transcription factors, was used as a target gene. One splicing variant of Osf2 mRNA was identified and its expression was restricted to the bones and osteosarcomas. The amount of the splicing variant of Osf2 mRNA was significantly higher in the blood of mice with metastasis than in the blood of the control group. The PCR-ELISA using Osf2 mRNA is a potential method to detect circulating osteosarcoma cells in peripheral blood.