The aim of the research was to ascertain different expression levels of miRNA-17-19b in different cell lines and to observe the changes of miR-17-19b expression in the cell line K562 with high expression level of miR-17-19b after 5-azacytidine (5-aza) treatment. Total RNA was extracted from K562, HL-60, NB-4 and HeLa cell lines, white blood cells of peripheral blood from patient with chronic myeloid leukemia (CML) and mobilized white blood cells of peripheral blood from normal persons, respectively. Total RNA was polyadenylated by poly (A) polymerases and the expressions of miR-17-19b in the cell lines and the above mentioned cells were detected by SYBR-green real-time PCR. The K562 cell line was treated with 2.5 µmol/L 5-aza for 24, 48 and 72 hours, then were collected at 96 hours. The changes of miR-17-19b expression were determined by real-time PCR after 5-aza treatment. K562 cell line proliferation was observed after inhibition of miR-19a function. The results showed that the expression levels of miRNA-17-19b in K562 cells and white blood cells of peripheral blood from CML patients were higher than those in mobilized white blood cells of peripheral blood from normal person. The expression level of miR-17-19b in K562 cells with high expression of miR-17-19b was down-regulated after 5-aza treatment. The proliferation of K562 cells was inhibited through suppression of miR-19a function. It is concluded that expression level of miR-17-19b is higher in K562 cell line and white blood cells of peripheral blood from CML patients than that in white blood cells of peripheral blood from normal person. Expression of miR-17-19b is inhibited in K562 cell line after 5-aza treatment. Inhibition of miR-19a in vitro can suppress the proliferation of K562 cell line.