To improve the bioactivity and increase the N-terminal homogeneity of a glucagon-like peptide-1 (GLP-1) analogue expressed in Pichia pastoris. The GLP-1 analogue. GGH, consisting of two tandem mutant GLP-1 (GLP-1[A2G]) fused with the N-terminus of human serum albumin (HSA), was expressed in P. pastoris. We also designed and expressed the novel GLP-1 analogue NGGH, which had a His-tag fused with the N-terminus of GGH and an enterokinase (EK) cleavage site at the fusion junction. The His-tag was removed by EK digestion to yield GGH(2), which was subsequently compared with GGH expressed in P. pastoris. The purification recovery of GGH(2) was 35 % compared with 23 % for GGH. Furthermore, the bioactivity of GGH(2) was 605 % higher than GGH, and N-terminal homogeneity was also improved. A simple method for the preparation of GGH(2) with a cleavable His-tag was developed, and the resultant protein possessed improved bioactivity and N-terminal homogeneity.