Objective · To optimize the method of the flow cytometry MoFlo Astrios EQ on single-cell sorting in 96-well plate. Methods · Using different aperture nozzles and sorting ways, the 32D, U937, iBMDM and 293T cells were used for single-cell sorting after the precise adjustment of the instrument and various parameters. The hole numbers with single cell and single-cell clones were detected after sorting. Results · In the single-cell sorting mode, the hole numbers with single cell were 83-9170 μm nozzle and 87-93100 μm nozzle. After 7-10 days of culture, the hole numbers with single-cell clones were 36-5870 μm nozzle. In 100μmnozzle, the hole numbers with single-cell clones were53-78electrostatic charge sorting and 69-81straight-down sorting, respectively. Conclusion · In single-cell sorting, a better cell viability and higher cloning rate are observed in 100 μm nozzle and straight-down sorting.