The invention discloses a 25-hydroxy-vitamin D3 detection kit, as well as a preparation method and applications thereof. The detection kit consists of a reagent I and a reagent II which are independent from each other, wherein the reagent I comprises the following components: a 25-hydroxy-vitamin D3-BSA conjugate, a biobuffer, a chelating agent, a surface active agent, a coagulant, a preservative, a stabilizer and water; the reagent II comprises the following components: rubber latex particles coated by a 25-hydroxy-vitamin D3 antibody, a biobuffer, a chelating agent, a surface active agent, a suspending agent, a preservative, a sealing agent, a stabilizer and water. The detection kit disclosed by the invention is high in detection sensitivity, high in specificity, accurate in quantification and wide in linear range, and is especially suitable for the detection of samples with extremely low 25-hydroxy-vitamin D3 content. In addition, the detection kit disclosed by the invention does not need to dilute the samples in advance in the detection process, facilitates the clinical use, and is easy to operate, low in detection cost and suitable for various full-automatic biochemical analyzers.