The invention discloses a micro-fluidic chip for whole blood detection, a detection device and a detection method thereof and belongs to the technical field of medical apparatuses. The micro-fluidic chip comprises a matrix, a sample adding groove formed in the matrix and at least one group of function units. The micro-fluidic chip is mounted on the detection device and is simple in structure, convenient to manufacture and suitable for market promotion. The micro-fluidic chip can integrate a process of separating whole blood to obtain blood serum, a process of mixing the blood serum and a diluent and a process of combining an antibody and an antigen specifically to a same chip and can detect a plurality of indexes on the same chip. The detection device and the detection method thereof provided by the invention do not need marks by means of an LSPR effect of nanoparticles in a testing process and detect the indexes without pollution but with high sensitivity in real time. The concentration of a to-be-tested object is judged by detecting the absorbance changing condition.