Casitas B-lineage lymphoma (CBL) is critical to the negative regulation of receptor protein tyrosine kinases (RTKs). Mutant CBL can act as an adaptor and gain function to cause myeloproliferative diseases. Currently, there is no beneficial treatment available for leukaemia patients with CBL mutations. Using phage-display technique, we identified a peptide inhibitor CBLock that binds a pocket in CBL’s tyrosine kinase binding domain (TKBD) in its native conformation as revealed by a crystal structure. CBLock inhibits proliferation, causes cell cycle arrest and apoptosis in leukaemia cells that harbour CBL mutations. CBLock disrupts the interaction between CBL mutants and RTK/adaptor protein, hindering the gain-of-function of CBL mutants. We find reduced tumour progression and improved survival upon conditional expression of CBLock in a xenograft AML model. CBLock also resulted in reduced FLT3 downstream signalling. Therefore, inhibiting CBL-TKBD in its native state presents a useful therapeutic opportunity in targeting mutant/inactive CBL-dependent leukaemia.