Glucagon-like peptide-1 (GLP-1) plays an important role in metabolic regulation, and its specifically modified peptide fragments are useful tools for studies on receptor-related signaling pathways. However, the expression of such peptides in Escherichia coli is often limited by inclusion body formation and low yield. In this study, a soluble expression and purification process for Arg34GLP-1(9-37) was established in E. coli. The coding sequence of Arg34GLP-1(9-37) was fused with different tags and an enterokinase cleavage site, and then cloned into different expression vectors. Recombinant expression was performed in Escherichia coli BL21 (DE3), and the soluble expression conditions of the fusion protein were optimized in a 5 L bioreactor. After purification by Ni2+-NTA affinity chromatography, the fusion protein was cleaved with enterokinase, and the target peptide was further purified by gel filtration chromatography. The results showed that the highest fusion protein yield reached (25.70 ± 0.52) g/L in LB medium when the pET-28(+) vector carrying a small ubiquitin-like modifier (SUMO) tag was used under the induction conditions of 28 °C, 2.0 mmol/L lactose, and 10 g/L MgSO4. During Ni2+-NTA affinity chromatography, impurity proteins were eluted with 50 mmol/L imidazole, while the target protein was eluted with 400 mmol/L imidazole, with a recovery rate of 92.30% ± 0.19%. After cleavage at 28 °C for 12 h, the cleavage rate reached 98.40% ± 0.18%. The target peptide Arg34GLP-1(9-37) was successfully obtained, and LC-MS analysis showed that its molecular mass was about 317 4.6 Da, which was consistent with the theoretical value. In conclusion, an efficient soluble expression and purification method for GLP-1-modified peptide fragments was established based on the E. coli expression system, providing a basis for the preparation and functional study of related peptides.