Abstract: The aim of this study was to construct the lentivirus expression vector of human FLT3-ITD mutation and to screen the AML cells for stable expression of FLT3-ITD. We screened the AML patient with FLT3-ITD mutation by peripheral blood DNA extraction and PCR, and then the ORF of FLT3-ITD was constructed using the method of homologous recombination, which used the BamH I and Not I double enzyme digestion of the ORF and the FIV vector. Results were confirmed by restriction enzyme identification and PCR. The constructed recombinant vector was co-transfected with VSVG and g/p into HEK293T cells to produce the lentiviral particles by transfection reagent. The lentiviral particles were transduced into K562 cells and the infection efficiency was measured by fluorescence microscope. The cells were sorted by the Flow Cytometer. The expression of FLT3 in the stable cell lines was detected by Western blot. There were 42 patients with FLT3-ITD mutation among the 207 patients we collected. We successfully amplified the ORF of FLT3 of the patient with highest mutant/wt ration. Then constructed the lentivirus expression vector FLT3-ITD/FIV and overexpressed it in K562 cell lines. We screened the cells expressed FLT3-ITD/FIV by fluorescence activated cell sorting, and Western Blot results proved the expression of FLT3-ITD. The over-expression 1entiviral vector of human FLT3-ITD/FIV has been successfully constructed and cell lines for stable expression of FLT3-ITD have been successfully established. This will shed a light on the future study of FLT3 function in AML and provide a new in vitro model for AML.