Genetic transformation is important for gene functional study and crops improvement. However, it is less effective in wheat. Here, we employed a multi-omic analysis strategy to uncover the transcriptional regulatory network (TRN) responsible for wheat regeneration. RNA-seq, ATAC-seq and CUT&Tag techniques were utilized to profile the transcriptional and chromatin dynamics during early regeneration from the scutellum of immature embryos in wheat variety Fielder. Our results demonstrate that the sequential expression of gene mediating cell fate transition during regeneration is induced by auxin, in coordination with changes in chromatin accessibility, H3K27me3 and H3K4me3 status. The built-up TRN driving wheat regeneration was found to be dominated by 446 key transcription factors (TFs). Further comparisons between wheat and Arabidopsis revealed distinct patterns of DNA binding with one finger (DOF) TFs in the two species. Experimental validations highlighted TaDOF5.6 (TraesCS6A02G274000) and TaDOF3.4 (TraesCS2B02G592600) as potential enhancers for the transformation efficiency in different wheat varieties.