SummaryIn contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)‐DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase‐mediated CUT&Tag (B‐CUT&Tag) to produce high‐quality libraries for interrogating TF‐DNA interactions. B‐CUT&Tag combines streptavidin‐biotin‐based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B‐CUT&Tag to probe genome‐wide DNA targets of Squamosa promoter‐binding‐like protein 9 (SPL9), a well‐established TF inArabidopsis; the resulting profiles were efficient and consistent in demonstrating its well‐established target genes in juvenile‐adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth‐defence trade‐offs. In addition, we established a method for applying qPCR after CUT&Tag (B‐CUT&Tag‐qPCR) and successfully validated the binding of SPL9 inArabidopsisand PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF‐chromatin interactions that is widely applicable to the annotation ofcis‐regulatory elements for crop improvement.