The invention relates to an improved body fluid color-developing fungi 1,3-beta-D-glucan detection kit, which comprises a reaction main-agent, a main agent combination solution, a sample treatment fluid, sterile water, a standard substance and a quality control material. With Tachypleus tridentatus Leach or Limulus polyphemus Linnaeus hemocyte lysate as a main raw material, the reaction main-agent contains G factor, B factor, C factor, clottable protein and a polypeptide chromogenic substrate. By a new preparation process, the reaction main-agent has characteristics of high stability and small intra-assay and inter-assay difference. The main agent combination solution adopts a new formula. After the main agent combination solution is mixed with the reaction main-agent, Tachypleus Amebocyte Lysate endotoxin reaction branch can be effectively shielded. According to the kit, rate-method enzyme kinetics detection is carried out by ELIASA. The detection speed is fast, and anti-interference performance is strong. In addition, the preparation technology is simple, and the product has stronger stability and higher specificity and sensitivity.