This study was aimed for the cloning of previously isolated transcription factor zinc finger gene (GaZnF) from Gossypium arboreum (desi cotton) and codonoptimized CEMB GTGene in plant expression vector (pCAMBIA-1301) and to study its expression in CIM-482 variety of Gossypium hirsutum for functional characterization under drought stress. A 531bp fragment of GaZnF gene was amplified through PCR from cDNA of drought stressed leaves of G. arboreum, after TA cloning was confirmed with previously reported sequence, GQ169757.1. Then it was cloned in pCAMBIA-1301 (plant expression vector) at Nco-I and Bgl-II sites in fusion with GUS exon. While GTGene was cloned by replacing hygromycin at Xho-I site and resulted vector was named as pGaZnF. The plant expression pGaZnF construct was transformed in Agrobacterium tumefaciens strain LBA 4404. The transient expression assay validated the construct by agroinfiltration of Nicotiana benthamiana leaves. The transformation efficiency of Gossypium hirsutum variety CIM-482 was 2.57% by using Agrobacterium mediated transformation system. The developmental and spatial expression analysis of GaZnF transgene was observed through Real time PCR in the stem and leaf of transgenic plants. The drought stressed transgenic plants showed highest expression: as 16 folds in leaves and 4.1in stem at vegetative stage while 14 folds in leaves and 3 in stem at mature plant stage. The amplification and integration of GaZnF transgene confirmed a fragment of 531bp through PCR and Southern blot analysis respectively. Expression of GaZnF gene was confirmed through ELISA and Western blot by using Anti-His tag polyclonal antibodies. Drought stressed transgenic plants confirmed a fragment of 95kDa (GaZnF-GUS fusion protein) in Western blot. While GTGene, through ELISA by using GTGene specific antibodies confirmed expression. The transgenic plants containing construct pGaZnF showed a steady increase in plant height after 10 day stress treatment. The mean increase in plant height, root shoot length ratio in T0 and T1 and percentage reduction in biomass, leaf to total plant weight ratio, shoot to total plant weight ratio and root to total plant weight ratio in the transgenic plants containing construct pGaZnF as compare to non-transgenic plants of T0. While leaf tissues from four months old drought stressed transgenic plants containing pGaZnF construct as compared to non-transgenic Gossypium hirsutum control plants, performed significantly for physiological parameters, including leaf relative water content (LRWC), photosynthetic rate (Pn), stomatal conductance (C), transpiration rate (E) and chlorophyll content in T0 and T1. Similarly, for biochemical analysis based on proline accumulation and total soluble sugars were significantly higher in drought stressed transgenic plants as compared to non-transgenic Gossypium hirsutum control plants in T0 and T1. Survival of transgenic plants in glyphosate spray also confirmed the integration and expression of GTGene successfully in cotton genome. Fluorescent in situ hybridization confirm single copy of GaZnF and GTGene on chromosomes of T2 progeny seeds. On the basis of this study it is concluded that the GaZnF is involved in activation of cascade of molecular network to regulate the drought stress tolerance in cotton. Thus the role of this gene in drought tolerance will not only provide direction for future engineering of drought-stress tolerance in cotton, but will also enable us to breed economically important cotton varieties.