Cotton, a key cash crop vital to the economy, suffers major yield loss due to Tobacco streak virus (TSV). TSV symptoms are frequently mistaken for physiological disorder, highlighting the need for accurate and timely diagnosis to enable effective disease control. Traditional diagnostics are time-consuming, relying on antibodies and specialized equipment. We developed a rapid, accurate RT-LAMP assay to diagnose TSV in cotton. The TSV inoculum was successfully maintained in Vigna unguiculata, Chenopodium amaranticolor and cotton in glasshouse. Molecular analysis through coat protein resulted in a 929 bp amplicon and the sequences were deposited in GenBank (PQ331227, PQ331228). The RT-LAMP assay employing a newly designed primer set efficiently amplified a 240 bp target region and yielded a distinct ladder-like banding pattern on gel electrophoresis confirming the virus. Colorimetric detection using hydroxy naphthol blue (HNB) dye allows visual identification of the virus with distinct color changes from violet to sky blue. A comparative sensitivity assay revealed that RT-LAMP has 10-fold higher sensitivity (0.05 pg/µl). These findings establish RT-LAMP as a rapid on-site diagnosis technique in comparison to RT-PCR for TSV diagnosis in cotton, facilitating early detection.