Tyrosine kinase inhibitors (TKIs), targeted therapeutic agents, have significantly improved survival outcomes in patients with chronic myeloid leukemia (CML). However, the emergence of drug resistance remains a challenge, with the underlying mechanisms still largely unknown. Deubiquitinating enzymes (DUBs) have been reported as potential targets in many human cancers. In this study, we identified ubiquitin‐specific protease 8 (USP8) as a potential prognostic target for CML drug resistance through bioinformatics analysis and validation in clinical samples. Functionally, knockdown of USP8 inhibited proliferation and increased apoptosis and TKI sensitivity in various CML cell lines. Mechanistically, immunoprecipitation‐mass spectrometry analysis and molecular docking demonstrated an interaction between USP8 and eukaryotic translation initiation factor 2 subunit alpha (EIF2S1). USP8 stabilizes EIF2S1 protein by inhibiting its K48‐linked ubiquitination, thereby preventing its degradation via the proteasome pathway. In other words, USP8 knockdown suppressed EIF2S1 protein expression and inhibited tumor growth both in vitro and in vivo , further suppressing TKI resistance. In summary, our results suggest that USP8 is overexpressed in CML and linked to resistance to TKIs. We have unveiled a previously unknown mechanism of CML drug resistance, which may provide novel perspectives on the advancement of targeted therapeutic strategies and clinical interventions.