The invention discloses a kit for determining glycosylated hemoglobin and a preparation method thereof. The kit comprises three liquid components a reagent R1, reagent R2 and a reagent R3 that are independently to each other. The reagent R1 consists of: a latex glycine buffer solution, polyethylene glycol-2000, Tween-80, glycerin, bovine serum albumin, and sodium azide, the reagent R2 consists of: a glycine buffer solution, an anti-IgG antibody, an anti-HbA1c antibody, sodium chloride, bovine serum albumin and sodium azide, and the reagent R3 consists of: a hemolysin diluent. The preparation method includes: preparing the reagents according to the component content; mixing a to-be-determined sample with the reagent 3, and using the hemolysin diluents to treat the to-be-determined sample; mixing the treated to-be-determined sample with the reagent R1 and reagent R2 and carry out full reaction; employing a fully automatic biochemical analyser to determine the absorbance difference after reaction; and calculating the concentration of glycosylated hemoglobin in the sample according to an absorbance change value. The kit provided by the invention has the advantages of high accuracy, and convenient operation, etc.