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Single cell analysis is of great significance to understand the physiological activity of organisms.Microfluidic droplet is an important analytical platform for single-cell analysis.1,2 We developed a device integrated with a flow-focusing structure and multi-step splitting structures to form 8-line droplets and encapsulate single cell in one droplet.With aqueous phase flow rate of 1 μL/min and the oil phase flow rate in a range of 0.5-5 μL/min,RSD of the droplet size in a single channel was 2%-5%,while in the 8 channels was 3%-6%.Droplet generation frequency reached 1021 Hz with the aqueous phase flow rate of 1.0 μL/min and the oil phase flow rate of 15 μL/min.Whole blood cells were encapsulated using the microfluidic multi-step droplet splitting system,while OptiprepTM diluents was chosen to improve the cell sedimentation phenomenon.A single-cell encapsulation efficiency of 31% was obtained with the theoretical blood cell concentration of 2.5×104/μL,and the multicellular droplet percentage was only 1.3%.It was demonstrated that the multi-step droplet splitting system for single cell encapsulation featured simple structure and high throughput.