Antibody single-chain variable fragments (scFvs) offer particular advantages over the full-size antibodies, including easy expression, efficient local concentration and fast body clearance. However, scFvs typically show low thermal stability that limits their biomedical and biotechnological applications. In this study, we examined the thermal stability of the human and murine vascular endothelial growth factor antibody scFv fragment by molecular dynamics simulations. A consistent observation was the dissociation of the light-chain (VL) and heavy-chain (VH) domains and loss of the native structures of both domains in the simulations at the elevated temperatures. The stability-limiting structural elements in the protein were revealed from the detailed analyses on the native contacts. We found that dissociation of the VL-VH domains was the first event leading to the unfolding of the native structure of the protein and the disruption of the VL-VH interface was largely due to the break of the interfacial hydrophobic and aromatic interactions while the hydrogen-bonding interaction between Gln38 in VL and Gln39 in VH remained. Within the β-barrel structure of the VL and VH domains, β-strands β6, β2 and β11 appeared to be the least stable. In addition, we found that the VH domain was more thermally resistant than the VL domain. Based on these findings, we discussed potential strategies to improve the stability of this therapeutically important scFv fragment.