The present invention relates to a thermostable alpha-1-antitrypsin mutein with a disulfide bond and a preparation method of the same, more precisely an alpha-1-antitrypsin mutein with improved thermostability, compared with the conventional thermostable alpha-1-antitrypsin mutein (Korean Patent No. 133252), resulted from the process of substituting the 168th and 189th amino acids of the alpha-1-antitrypsin with cysteines which are then linked by a disulfide bond, with keeping its alpha-1-antitrypsin activity unchanged, and a preparing method of the same. The thermostable alpha-1-antitrypsin mutein with a disulfide bond of the invention can be effectively used for a protein based therapeutic agent which is very stable in vivo, and can be applied further in various fields including in the development of a diagnostic reagent and in the preparation of an affinity column.