Fibroblast growth factor 2 (FGF2) plays an important role in multiple physiological functions such as tissue repair. However, FGF2 has a short half-life in vivo due to protease degradation, thus limiting its clinical application. Traditional PEGylation has typically focused on the N-terminal α-amino group of FGF2. These modifications do not consider potential effects on protein function or structure, and sometimes lead to decreased bioactivity. In this study, we generated three PEGylated FGF2 variants based on the structure of the FGF2-FGFR-heparin ternary complex via gene mutation and PEGylation, and investigated the effects of these PEGylated sites on protein stability and bioactivity. Compared with native FGF2, all PEG-FGF2 conjugates exhibited significantly improved stability. Conjugates PEGylated at a site separated from both binding regions more effectively promoted proliferation, migration and angiogenesis than FGF2 in vitro, and exhibited excellent wound healing activity in vivo, making these conjugates potential therapeutic candidates for wound healing. Computer-assisted modification based on structure reveals the detailed structural characteristics of proteins, allowing efficient protein modification for improved stability and activity. This structure-guided PEGylation offers a more reliable modification strategy and should be applied for the rational design of protein-based therapeutics.