Objective: To establish a program for screening monoclonal cell lines with high expression of fluorescent protein after stable transfection using a microplate imaging system. Methods: HBE cells were stably transfected with GFP (green fluorescent protein) by lentivirus and cells with high fluorescence intensity were seeded into a 96-well black plate (with clear bottom) by flow cytometric sorting. Single cells were seeded into all wells, except one well, into which 50 cells were seeded to acquire an appropriate setting during imaging. According to the screening protocol, the imaging and analysis functions of the microplate imaging system were employed four times to perform screening across 95 wells. Wells containing one single cell were picked out on the day of sorting and wells containing cell colonies were picked out after clone formation. After cells in the selected wells were individually disassociated and cultured in new wells, wells containing cells with high fluorescence intensity were picked out. After achieving 4-8× population expansion, wells containing cells exhibiting normal proliferative capacity were picked out. The fluorescence intensities of selected monoclonal cell lines were ultimately confirmed by flow cytometry and fluorescence imaging. Results: According to the screening program, 89 wells containing one single cell, 19 wells containing cell colonies, 6 wells containing cells with high fluorescence intensity and 3 wells containing cells exhibiting normal proliferative capacity were picked out sequentially. The selected cell lines from those 3 wells exhibited significantly higher mean fluorescence intensities and higher percentages of cells with strong fluorescence, compared to parental cells. Conclusion: Application of a microplate imaging system to the screening of monoclonal cell lines with high expression of fluorescent protein after stable transfection, based on the screening program, is simple and convenient (compared with traditional microscopy-based observation/imaging), showing considerable potential for widespread application.