Objective：To establish a workflow using a microplate imaging system for screening monoclonal cell strains with high fluorescent protein expression after stable transfection. Methods：Human bronchial epithelial（HBE）cells were transfected with green fluorescent protein（GFP）gene using a lentiviral vector. Single cells with strong fluorescence were sorted by flow cytometry into a 96-well plate. According to the established screening protocol，four rounds of stepwise screening were performed using the imaging and analysis functions of the microplate imaging system：on the day of sorting，wells containing a single cell were identified；when the majority of clonal clusters contained more than 20 cells，wells with clonal clusters were selected；after trypsinization and transfer of cells from the selected wells to new wells，wells with cells exhibiting higher mean fluorescence intensity were identified；when rapidly proliferating cells expanded 4-8 fold，wells with cells exhibiting normal proliferation were selected. The stably transfected cells were continuously maintained under the same culture conditions as the monoclonal cells. When the monoclonal cells proliferated to an adequate number，flow cytometry and fluorescence imaging were used to compare the fluorescence intensity between the finally selected monoclonal cell strains and the stably transfected cells. Results：Following the screening protocol，89 wells containing a singlecell，19 wells with clonal clusters，6 wells with cells exhibiting higher mean fluorescence intensity，and 3 wells with normally proliferating cells were successively identified. Flow cytometric analysis demonstrated that the monoclonal cell strains from these three wells exhibited an approximately 9-fold increase in mean fluorescence intensity and a significant increase in the percentage of cells exhibiting strong GFP fluorescence，compared to the stably transfected cells. This improvement was corroborated by microplate imaging，which revealed markedly superior GFP fluorescence in the monoclonal cell strains. Conclusion：Using a microplate imaging system in combination with the established screening protocol enables stepwise and efficient screening of monoclonal cell strains with high fluorescent protein expression after stable transfection. Compared to traditional microscopy or other imaging methods，this approach is simpler，more convenient，and holds significant potential for broader application.