Background and Purpose Fibroblast growth factor 2 (FGF2) plays an important role in multiple physiological functions such as tissue repair. However, it has a short half-life in vivo, thus limiting its clinical application. Experimental Approach Based on the crystal structure of FGF2, surface-exposed sites near the receptor and heparin binding region or separated from both binding regions, were selected and PEGylated to investigate the effects of these sites on protein stability and bioactivity. The efficacy of FGF2 conjugates for wound healing in vitro was screened by cell proliferation, migration of human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs). Angiogenesis activity was assessed by tube forming and aortic ring assays. The stability was confirmed in plasma and wound fluid. The in vivo wound healing of FGF2 conjugates were further evaluated on a cutaneous wound model using H&E, IHC, IF, and collagen staining. Key Results Compared with native FGF2, all PEG-FGF2 conjugates exhibited significantly improved stability in plasma and wound fluid. Compound 6 more effectively promoted proliferation and migration in HDFs and HUVECs than FGF2, and exhibited excellent angiogenesis and wound healing activity in vivo. However, conjugates with the PEGylated sites near the receptor and heparin binding regions showed some reduction in bioactivity, with a greater loss of bioactivity for protein with site near the heparin binding domain. Conclusion and Implications Heparin binding domain may be a key contribution region to activity of FGF2. Compound 6 is a potential therapeutic candidate for wound healing, deserving further investigation.