The invention provides a micro-fluidic chip for chemiluminescence detection, and a using method and a reagent cleaning method thereof, and belongs to the field of biochemical testing devices. The micro-fluidic chip comprises a substrate and a cover plate which form a sealing environment and are used for a micro-fluid to flow, the substrate is covered with the cover plate, and the substrate comprises: a sample adding hole and a diluted solution adding hole at the first layer; a sample quantification groove, a sample overflow groove, a diluted solution quantification groove and a dilution overflow groove at the second layer; an incubation groove at the third layer, solution storage grooves at the fourth layer, a detection groove at the fifth layer and a waste liquid groove at the sixth layer, wherein the sample quantification groove is communicated with the incubation groove, the diluted solution quantification groove is communicated with the incubation groove, the incubation groove is communicated with the detection groove, and the detection groove is communicated with the waste liquid groove through a hydrophobic flow channel. The separation, mixing and detection of a liquid is realized without repeatedly adding a sample, and the whole process is automatically completed, so the efficiency is increased, and the cost is realized; and the cleaning of a reaction reagent is realized, so the working efficiency is increased.