The purpose of this study was to establish a SYBR Green I real-time quantitative RT-PCR method for investigating the correlation between CML28 mRNA expression levels and relapse of leukemia after allo-hematopoietic stem cell transplantation (HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitoring of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph(+) ALL. The results showed that the sensitivity of the established method was at 10(-4) (0.05 ng) level, with interassay variation and intraassay variation of standard samples both below 10%. The CML28 was highly expressed in AML and CML-BP or AP. CML28 level in newly diagnosed group was (6.58 +/- 2.34) x 10(-2), in pre-conditioning regimen group was (2.19 +/- 0.32) x 10(-2), in group that 1 month after HSCT was (1.35 +/- 1.28) x 10(-2), in group that 3 months after HSCT was (4.57 +/- 6.39) x 10(-3). CML28 can be detected 3 months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2 x 10(-2)) survived without relapse, the other 2 case with high level (>2 x 10(-2)) relapsed within one year, 1 case died and 1 case received the second time HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2 x 10(-2) and relapse has taken place. The conclusions was made that CML28 mRNA level is obviously correlated with the development of leukemia. Serial quantification of CML28 mRNA levels are necessary for HSCT recipients, and more informative than a single detection. Using of this assay to evaluate MRD in the patients received HSCT is helpful for prediction of relapse.