Accumulation of anthocyanin pigments in plants not only involved in imparting the colour in different plant parts but also acts as a great osmoregulator. The increase in turgor pressure through positive osmoregulation can leads towards improvement in fiber characteristics of cotton. Based on this fact, an effort was made in the current study to improve fiber in local cotton variety by transforming flavonoid genes dihydroflavonol 4- reductase (DFR) & Flavonoid 3’5’ hydoxylase (F3’5’H). The DFR is an active enzyme of the flavonoid pathway and highly substrate specific. Protein docking analysis revealed that proline rich region, amino acids at positions 12, 26 and 132-157 in Iris as well as Gossypium based DFRs were not involved in determining substrate preference but a play role in substrate attachment and anthocyanin production. The F3’5’H enzyme is known for synthesis of 3’, 5’- hydroxylated anthocyanins. Protein docking results showed the best binding energies of Viola F3’5’H with ligands i-e - 7.6 (naringenin) & -8.3 (quercetin), revealing its greater capability to reduce substrates and produce anthocyanins as compared to Gossypium F3’5’H which has binding affinities -7.9 (naringenin) and -7.4 (quercetin). Plant expression vector pCAMBIA-1301 was constructed with F3’5’H and DFR genes for cotton transformation. The excision of 4032 bp and 11000 bp bands from pCAMBIA-F3’5’+DFR through restriction digestion with KpnI and XbaI enzyme confirmed successful ligation of both genes in plant expression vector. After the confirmation of F3’5’H and DFR genes ligation in pCAMBIA1301, the recombinant plasmid (pCAMBIAF3’5’+DFR) was electroporated in Agrobacterium (LBA4404) cells by using electroporation device. The amplification of 476 bp and 537 bp through Agrobacterium colony PCR revealed introduction of recombinant plasmid in Agrobacterium. The cotton variety, VH-319 embryos were subjected to inoculation with Agrobacterium containing both genes and the cotton plantlets developed from the embryos were subjected to confirmation of transgenes. Amplified products of 476 bp and 537 bp from extracted genomic DNA confirmed successful integration of transgenes in cotton plants. Further signal obtained through hybridization of gene specific probe on nitrocellulose membrane in DNA dot blot assay also validated the presence of both genes in transgenic cotton plants. Overall transformation efficiency was calculated to be 2.1%. The mRNA expression level of F3’5’H and DFR genes was measured to be 1.0-5.3 and 1-4 fold higher in leaves and 1-3 fold higher in fiber of transgenic cotton plants respectively as compared to non-transgenic control cotton plants through quantitative Real Time PCR. Similarly, gene integration revealed single copy number of transgene F3’5’H and DFR on chromosome number 16 when subjected to fluorescent in situ hybridization (FISH) and its Karyotyping. Quantitative estimation of anthocyanin contents in transgenic cotton lines was undertaken by pH differential method. Maximum obtained anthocyanin concentration was in range of 1.79 µg/g to 1.0 µg/g. The anthocyanins produced in transgenic cotton plants, though did not impart any phenotypic change but have shown a positive impact on other physical properties of fiber particularly length and strength. Fiber data analysis showed significant improvement in staple length which was found to be increased from 26.3 mm to 31.6 mm (20.1%), fiber strength ~ 23.8 to 32.4 g/tex (32.7%), uniformity index ~ 82-86 (5.2%) and the micronaire value was found to be improved from 4 to 3.2 µg in transgenic cotton plants. Electron microscopic examination showed that transgenic cotton fibers possess greater number of twists in addition to smooth and compact surfaces as compared to non transgenic control cotton plant. A positive correlation of transgene was found with physiology of transgenic cotton plants like maximum photosynthetic and evaporation rate along gaseous exchange in transgenic cotton plant which was recorded to be 6.5 µmol/m2/s, 6.55 mmol/m2/s and 154 mmol/m2/s respectively as compared to 3.2 µmol/m2/s, 1.67 mmol/m2/s and 54 mmol/m2/s in non transgenic cotton plants. Morphological traits like plant height were found as independent factor with respect to monopodial and sympodial branches. Two other key characters i-e boll and lint weight showed positive significant correlation according to Pearson correlation. The study resulted in provision of unique information for better utilization of this trait in molecular breeding program which in combination with other fiber trait will provide a great breakthrough to cotton growers and to textile industry in specific for saving their import losses.