The objective of this study was isolation of humanized single-chain variable fragment (scFv) antibodies against IL1RAP, a promising cancer marker in leukemia and other tumors. Here, we screened for antibody fragments that recognized IL1RAP using a phage-display library of humanized scFv through several rounds of bio-panning and further FACS selection of enriched yeast-display sub-library. Two specific scFv clones 02 and 61 were isolated with high affinity against IL1RAP. Selected IL1RAP scFv antibodies containing Fc segment were expressed and purified from 293F cells. scFv-02 expressed and purified from mammalian cells exhibited higher IL1RAP binding affinity on IL1RAP-positive 293F cell surface compared to scFv-61. In addition, scFv-02 was significantly specific to IL1RAP, as no binding signal of IL1RAP was detected on the surface of IL1RAP CRISPR-Cas9 edited cell line generated in this study. Two scFv antibodies targeting IL1RAP were isolated by phage and yeast display screening. scFv-02 could serve as a promising material for further development of IL1RAP targeted immunotherapy of leukemia and other diseases. Our work also modified the process for the selection of scFv against targeted antigen by stepwise using phage display, yeast display and mammalian cell display techniques.