BackgroundDFRis a crucial structural gene in plant flavonoid and polyphenol metabolism, andDFRknockout (DFR-KO) plants may have increased biomass accumulation. It is uncertain whetherDFR-KO has comparable effects in tobacco and what the molecular mechanism is. We employed the CRISPR/Cas9 method to generate a knockout homozygous construct and collected samples from various developmental phases for transcriptome and metabolome detection and analysis.ResultsDFR-KO turned tobacco blossoms white on homozygous tobacco (Nicotiana tabacum) plants with bothNtDFR1andNtDFR2knockout. RNA-seq investigation of anthesis leaf (LF), anthesis flower (FF), mature leaf (LM), and mature root (RM) variations in wild-type (CK) andDFR-KO lines revealed 2898, 276, 311, and 101 differentially expressed genes (DEGs), respectively.DFR-KO primarily affected leaves during anthesis. According to KEGG and GSEA studies,DFR-KO lines upregulated photosynthetic pathway carbon fixation and downregulated photosystem I and II genes.DFR-KO may diminish tobacco anthesis leaf photosynthetic light reaction but boost dark reaction carbon fixation.DFR-KO lowered the expression of pathway-related genes in LF, such as oxidative phosphorylation and proteasome, while boosting those in the plant–pathogen interaction and MAPK signaling pathways, indicating that it may increase biological stress resistance.DFR-KO greatly boosted the expression of other structural genes involved in phenylpropanoid production in FF, which may account for metabolite accumulation. The metabolome showed that LF overexpressed 8 flavonoid metabolites and FF downregulated 24 flavone metabolites. InDFR-KO LF, proteasome-related genes downregulated 16 amino acid metabolites and reduced free amino acids. Furthermore, the DEG analysis on LM revealed that the impact ofDFR-KO on tobacco growth may progressively diminish with time.ConclusionThe broad impact ofDFR-KO on different phases and organs of tobacco development was thoroughly and methodically investigated in this research.DFR-KO decreased catabolism and photosynthetic light reactions in leaves during the flowering stage while increasing carbon fixation and disease resistance pathways. However, the impact ofDFR-KO on tobacco growth steadily declined as it grew and matured, and transcriptional and metabolic modifications were consistent. This work offers a fresh insight and theoretical foundation for tobacco breeding and the development of gene-edited strains.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04307-7.