The invention discloses a lactic dehydrogenase detection kit and a preparation method of the kit. The kit disclosed by the invention consists of a reagent I and a reagent II which are independent each other, wherein the components of the reagent I comprise a biobuffer, a metal ion complex, a lactic dehydrogenase reactive substrate, a surfactant, a lactic dehydrogenase activity activating agent, a preservative and water; and the components of the reagent II comprise a biobuffer, a nicotinamide adenine dinucleotide oxidation state, a surfactant, a preservative and water. The detection kit disclosed by the invention adopts a dual reagent mode to effectively avoid the interference of the nonspecific reaction and synchronously furthest reduce the interference of the sample turbidity, thereby guaranteeing the stable and reliable measurement result; and the detection kit has the advantages of good stability, high precision, wide linear testing range, good repeatability, and strong anti-interference performance and the like. In addition, the detection kit disclosed by the invention does not need to pre-dilute the sample in the detecting process, thereby being convenient for clinical use, simple and fast to operate, and suitable for an automatic biochemical analyzer.