The invention discloses a method and a device for detecting and separating HP ELISA. According to the method, a prepared magnetic bead provided with HP, an enzyme-labeled antibody, an enzyme reaction substrate, a gastric juice sample and a cleaning fluid are placed in different liquid storage tanks on a micro-fluidic chip by the aid of an electric fluid force, and flow of liquids in the different liquid storage tanks is electrically controlled , so that ELISA is fully and automatically detected; and a detected fluorescence signal is taken as a trigger signal, so that an HP sample is fully and automatically separated. The device comprises a platform structure, the micro-fluidic chip and an electric cabinet, wherein the micro-fluidic chip comprises a glass sheet, a PDMS (polydimethylsiloxane) square plate and a driving electrode, a micro channel, a mixed channel and a detection area are formed in the surface of the PDMS square plate in a photoetching manner by the aid of a soft lithography technology, and the liquid storage tanks are arranged on the PDMS square plate. The device for detecting and separating the HP ELISA has the advantages as follows: the device is small in size, light in weight, convenient to carry and operate and low in construction cost and can be handheld for field detection, thereby facilitating popularization and application.