The invention discloses a microfluidic assay chip integrated capillary electrophoresis separation with chemiluminescence detection. The microfluidic chip consists of a buffer solution storage tank, a sample liquid storage tank, a sample waste liquid tank, a waste liquid tank, a sheath flow storage tank, a sample introduction channel, a separation channel, a sheath flow channel and a detection channel. A terminal of the separation channel and a starting point of the detection channel are communicated with the sheath flow channel. A chemiluminescence reagent enters the detection channel through the sheath flow channel; and when compositions to be assayed after the capillary electrophoresis separation of the chip meets the chemiluminescence reagent at a connecting point of the separation channel and the detection channel, the compositions are mixed with chemiluminescence reagent in the detection channel and then undergo luminous reaction. The inside of the separation channel is provided with a microporous plug to prevent the chemiluminescence reagent from flowing backwards into the separation channel under the driving of pressure difference so as not to influence electrophoresis separation. The microfluidic assay chip has the characteristics of high separation efficiency, high detection sensitivity, simple structure, small volume, light weight, convenient operation and the like, and is ideal to prepare a portable micro total separation system.