A prothrombin time test kit consists of thromboplastin and a buffer liquid system, wherein the thromboplastin is rabbit brain tissue factors and recombinant tissue factors. The buffer liquid system comprises 30-50mM Tris-HCL, 3-10% of glycine, 1-5% of bovine serum albumin, 0.6-1.3% of sodium chloride, 6.8-8.4mM of calcium chloride and 30.5% of NaN, and the pH value of the buffer liquid system is 6.0-8.0. The thromboplastin also comprises rabbit cephalin. According to the invention, the defects that the sensitiveness indexes of various instruments are different due to the differences of the methods of the different instruments can be overcome, the high-sensitivity human recombinant thromboplastin and low-sensitivity rabbit brain powder extracting thromboplastin are combined, the PT reagent capable of meeting different methods of coagulometer can be prepared according to certain proportion, and the ISI (insulin sensitivity index) value is close to 1.0. The invention also discloses a preparation method of the prothrombin time test kit simultaneously.