The invention discloses a microfluidic paper chip and a chemiluminescence immunoassay method. A sampling layer, a detection layer, a water-absorbing layer and a rotating shaft are assembled into the microfluidic paper chip, wherein the sampling layer and the water-absorbing layer can be rotated around respective spindles so as to align with the detection layer and form a hydrophilic channel. In a detection operation process, a buffer solution and a developing solution for washing separation and chemiluminescence measuring sampling are uniformly dropwise added at a sampling port of the sampling layer, so that the complex operation of separately dropwise adding is reduced; an antigen specimen and a detection sample can be respectively dropwise added into the reaction tanks on the detection layer, so that a standard curve can be obtained on a same paper chip after detection and the concentration of the detection sample can be calculated; the microfluidic paper chip and the chemiluminescence immunoassay method disclosed by the invention can be used for reducing the complex operation in the detection process, effectively eliminating error and increasing the detection accuracy.