The objective of this study was to investigate the specificity of detecting the phosphotyrosine level with anti-phosphotyrosine monoclonal antibody PY20 for diagnosis and prognosis of patients with chronic myeloid leukemia (CML) and the possibility of its clinical application. The positive rate of PY20 in 28 newly diagnosed CML patients was detected by flow cytometry using anti-PY20 antibody, the bcr-abl fusion gene was detected by nested RT-PCR, the Ph chromosome was measured by R-banding cytogenetic analysis, and the coincidence of PY20 positive rate with results of bcr-abl fusion gene and Ph chromosome detection was compared. In addition, the positive rate of PY20, the changes of bcr-abl fusion gene and Ph chromosome were determined in follow up 7 CML patients after allo-hematopoietic stem cell transplantation. The results indicated that the positive rates of PY20 in 28 newly diagnosed CML patients in groups of chronic phase (CP), accelerated phase (AP), and blast phase (BP) were (40.31% +/- 1.22)%, (77.28 +/- 1.14)% and (78.12 +/- 1.32)% respectively. The positive rate of PY20 in CP was lower than that in AP and BP (p < 0.05). There was no difference in positive rate of PY20 between AP and BP (p > 0.05). PY20 expression level of leukocytes from peripheral blood and bone marrow showed no difference (p < 0.05). The positive rates of PY20 in patients with CR, PR and NR were (15.56% +/- 1.51)%, (38.73% +/- 2.31)% and (60.43% +/- 2.04)% respectively. The positive and negative coincidence between PY20 and RT-PCR was 92.31% and 95.45% respectively. The positive and negative coincidence between PY20 and Ph Chromosome in newly diagnosed patients was 88.46% and 95.46% respectively. Ph chromosome and PY20 were all negative in 7 CML patients after allo-HSCT. Bcr-abl fusion gene was negative persistently in 5 patients, but in the other 2 patients, the fusion gene was persistently positive. In conclusion, the detection of the level of phosphotyrosine in CML cells has high sensitivity and specificity. The results of PY20 cell positive rate combined with detection of bcr/abl fusion gene and Ph chromosome might be useful in diagnosis as a good index of monitoring.