The invention relates to a high-density lipoprotein cholesterol detection kit and a use method thereof, relating to the biochemical detection field. The high-density lipoprotein cholesterol detection kit is high in stability, low in toxicity and high in accuracy and precision. The kit contains a kit 1 and a kit 2, wherein the kit 1 contains a surfactant, polyethylene sulfate, lauryl sodium sulfate, a compound stabilizer, calcium chloride, cholesterol oxidase, cholesterol esterase, ascorbic acid oxidase, catalase, 4-amino antipyrine, proclin series preservatives, glycol, saccharose and the balance of a buffer solution; and the kit 2 contains a surfactant, polyoxyethylene alkyl tribenzy, glycol, saccharose, peroxidase and the like. Compared with polyanions adopted in a traditional method, the kit contains multiple mixed surfactants including poloxamer series surfactants, polyethylene sulfate, lauryl sodium sulfate and the like and has relatively good specificity and selectivity.