The invention discloses an integrated electrochemical light emitting paper micro-fluidic chip, a preparation method and a method using the chip to detect hepatitis B surface antigen. The preparation method of the paper micro-fluidic chip comprises the following steps of preparing an electrode layer; preparing a paper channel layer on paper, immobilizing antibodies in an antibody embedding area and a detection area, and covering the surface of a sample area with a blood filtering film, so as to functionalize the paper channel layer; using a hot press technique to prepare the electrochemical light emitting paper micro-fluidic chip which comprises an upper sealing layer, the paper channel layer, the electrode layer and a lower sealing layer from top to bottom. The method using the chip to detect the hepatitis B surface antigen comprises the following steps of dripping a full blood sample into a sample adding area, enabling the sample to generate immune reaction in the flowing process, cleaning, and putting into an electrochemical light emitting detector for detection. The integrated electrochemical light emitting paper micro-fluidic chip has the advantages that the functions are rich, the detection sensitivity is high, the use is simple and convenient, and the cost is low; the sample pretreatment, mixing, reaction and detection are integrated, and the chip can be widely applied to the field of medical detection.