The plant transient expression system plays an important role in analysis of subcellular localization, interaction and promoter activity of protein. Here, we established a transient expression system in cotton cotyledons based on Agrobacterium-mediated transformation with GUS and eGFP as reporter genes. We investigated the optimal conditions for the system, including cotyledon growth time, Agrobacterium concentration, injection volume and co-culture time. The highest efficiency of exogenous gene expression was 50 μL Agrobacterium LBA4404 or GV3101 with OD₆₀₀=0.5 and co-culture for 2–3 days in cotton cotyledons germinated for 6 days. The transient expression system is convenient and only needs 8 to 13 days to detect exogenous protein expression from germination to results. The system may have a good application for analysis of gene function in cotton.