Exendin-4 is a glucagon-like polypeptide-1 (GLP-1) analog derived from lizard venom, but its short half-life affects drug administration compliance. An anti-HSA nanobody with a smaller size to guide the peptide coupling to Human Serum Albumin(HSA) in vivo may be a feasible strategy for constructing inexpensive, long-acting exendin-4 analogs. For this purpose, a fusion protein (exendin-4-(G4S)3-sdAbHSA), in which a humanized anti-HSA nanobody to the C-terminal of exendin-4 through the (Gly₄Ser)₃ flexible joint, was constructed. The target gene was designed according to the preferred codons and cloned into expression vector pET21b of Escherichia coli. The fusion protein could be efficiently expressed as a soluble protein, and purified to a purity over 98% by two steps of chromatography columns. In the streptozotocin-induced mouse diabetes model, the purified product had similar hypoglycemic activity as exendin-4, but dropped to the lowest value from 1 to 2 h to more than 8–10 h. The results show that this construction form does not interfere with the binding of exendin-4 to GLP-1 receptor, and can significantly prolong its half-life in vivo. This study has important reference value for constructing long-acting exendin-4 analogs and establishing efficient and green production process.