Abstract: Objective：To compare the sorted single cell rate and cell viability with different diameters of nozzles by the thirdgeneration flow cytometry sorter FACS Aria Ⅲ so as to further improve the optimization of parameter settings and conditions for singlecell microplate sorting. Methods：Singlecell sorting of four different types of cell linesRaji cells, A549, DT40, RAW 264.7 cell linewas done on FACSAria Ⅲ flow cytometry sorter and 96well microplate. 70 μm, 100 μm and 130 μm diameter nozzles were adopted respectively, and the sorted single cell rate and the cell colonyforming rate were counted. Results： In the singlecell sorting mode, the percentages of sorted single cell with three nozzles were from 86.55% to 93.44%, which meant that the hole numbers with single cell in the 96well micorplate were from 84 to 92. After 7day culture, the single cell clone formation rate were 36.90%57.78%, 57.65%76.09% and 67.44%86.67%, respectively. Single cell clone hole numbers sorted by 70 μm, 100 μm and 130 μm diameter nozzles were 3152, 4970 and 5878, respectively. Conclusions：In single cell sorting with the microplate and under the premise of accurate adjustment and optimization of parameters, 100 μm or 130 μm nozzles should be given preference for single cell activity.