Objective To study the cytotoxicity of bi-chimeric antigen receptors modified T lymphocytes (BiCAR-T) on the human acute myeloid leukemia (AML) cell line HL60 in vitro and the anti-tumor effects of BiCAR-T on the NOD SCID mouse model of AML in vivo.Methods The BiCAR-T were prepared and the expression of chimeric antigen receptor (CAR) of prepared BiCAR-T was analyzed by flow cytometry.In vitro study was divided into two groups:the experiment group (BiCAR-T) and the control group (T lymphocyte).The killing rate of BiCAR-T in vitro on HL60 cells was determined by CCK8 assay and the level of interferon-γ (IFN-γ) secreted from BiCAR-T co-culturing with HL60 cells for 48 hours was detected by enzyme linked immunosorbent assay (ELISA) at different effect/target ratios (5∶1,10 ∶ 1,20 ∶ 1).The NOD SCID mice AML model was established by the injection of HL60 cells through tail vein and used to assess the antitumor effects in vivo.The mice were randomly divided into three groups according to the random number table:the blank control group receiving 0.9％ NaCl 0.2 ml through tail vein,the model group and the treatment group receiving 1 × 107 HL60 cells in 0.2 ml phosphate buffer saline (PBS).After 20 days,the treatment group was injected with 2 × 107BiCAR-T in 0.2 ml PBS 3 times a week for 2 weeks,while the other two groups received 0.9％ NaCl 0.2 ml.The pathological changes in the mice livers and spleens were observed after 2 weeks of treatment.Results The CAR expression rates of BiCAR-T were more than 50.00％.In vitro experiments proved that the killing rates of BiCAR-T in the experimental group and T lymphocytes in the control group on HL60 cells were (25.43 ±1.32)％ vs.(16.18 ±0.75)％,(50.33±3.11)％ vs.(25.47±1.27)％,and (85.89 ± 3.96) ％ vs.(49.45 ± 2.77) ％ at different effect/target ratios (5 ∶ 1,10 ∶ 1,20 ∶ 1).The killing efficiency of BiCAR-T and T lymphocytes on HL60 cells was significantly different (F =404.17,P ＜ 0.001);the killing efficiency of BiCAR-T and T lymphocytes on HL60 cells was significantly different at different effect/ target ratios (F =548.09,P ＜ 0.001);and the killing efficiency on HL60 cells in the experimental group (BiCAR-T) was significantly higher than that in the control group (T lymphocytes) at different effect/target ratios (F =45.36,P ＜ 0.001).The IFN-γlevels secreted from BiCAR-T in the experiment group and T lymphocytes in the control group co-culturing with HL60 ceils after 48 h were (435.65 ± 20.44) pg/ml vs.(356.75 ± 19.87) pg/ml,(1 639.98 ± 95.75) pg/ml vs.(1 109.37 ± 80.98) pg/ml,and (3 467.43 ± 187.54)pg/ml vs.(2 245.52 ± 112.66)pg/ml.The IFN-γlevel in the experiment group (BiCAR-T) and the control group (T lymphocytes) was significantly different (F =156.24,P ＜ 0.001);the IFN-γ level was significantly different at different effect/target ratios (F =857.67,P ＜ 0.001);the IFN-γlevel in the experimental group (BiCAR-T) was significantly higher than that in the control group (T lymphocytes) at different effect/ target ratios of 5 ∶ 1,10 ∶ 1,20 ∶ 1,respectively (F =46.31,P ＜ 0.001).The result of hematoxylineosin staining (HE) staining showed that leukocyte infiltration in the treatment group was significantly decreased compared with the model group.Conclusion The experimental results showed that BiCAR-T is a kind of efficient targeted immunocyte modified by gene engineering,and it can significantly inhibit leukocyte infiltration of AML in vivo and in vitro.