Populus tomentosa Carr. is a native species in section Leuce with fast-growing and high wood quality in northern China. However, its catkin pollution brings a lot of inconvenience to people’s life and production activity. To obtain sterile poplar, one sterility construct 35S-PtAP3-IR-NOS was transformed into P. tomentosa. The positive results verified by PCR included twelve 35S-PtAP3-IR-NOS transgenic poplar plants. RT-qPCR showed little difference in expression among transgenic lines. The highest level of expression was 4.96 times and the lowest was 3.02 times of that of the reference gene. The expression of upstream gene LFY and PI in transgenic plants relatively declined compared with the wild type, there were no dramatic changes of downstream gene SEP2,SEP3 and SEP4 between transgenic poplar plants and wide type control. Our research suggests that the expression of AP3，LFY and PI in transgenic poplar plants is obviously suppressed, which provides reference for genetically engineered sterility.