The invention relates to a method for continuously enriching and separating phosphoeptide at high throughput. The method comprises the following steps: assembling a microfluidic device; cleaning the microfluidic device for 1 to 3 minutes with two kinds of cleaning solution in the staying time of between 30 and 60 seconds in sequence by using a syringe pump as a fluid transportation power source, and feeding proteolysis products to the microchannel of the device; and enriching, cleaning and eluting to prepare the enriched product. The separation method has simple operation, and can regulate and control the stay time of the fluid in the microchannel by regulating the pushing speed of the syringe pump, namely automatically control the enriching condition by regulating the contact time of thefluid with adsorbing materials, thereby avoiding repeated manual operation required by the conventional methods, and realizing continuous, quick and high-throughput separation and enrichment of the phosphoeptide by means of inherent advantages of the microfluidic device.