Abstract: The present invention relates to human insulin expression plasmids and a method for producing insulin using the same. The plasmids comprise a sequence encoding a compound of the formula R—B—X-A, in which R is a leader peptide of the formula of Met-Thr-Met-Ile-Thr-Y (SEQ ID NO: 36), in which Y is one selected from lysine, arginine, a peptide containing lysine as an amino acid at its C-terminal, or a peptide containing arginine as an amino acid at its C-terminal; B is human insulin B-chain or analogue thereof; X is a peptide connecting B with A; and A is human insulin A-chain or analogue thereof. The method for preparing insulin using the plasmids according to the present invention converts the proinsulin fusion protein into human insulin in a single enzymatic cleavage process and minimizes the generation of by-products after the enzymatic cleavage, thereby producing insulin at a high yield.