In vitro aggregated single-chain Fv antibody (scFv) was analyzed by attenuated total reflectance Fourier transform infrared spectra and scanning electron microscope images. The spectroscopic results demonstrate that the formation of beta-strands is a first trigger to the off-pathway toward insoluble aggregates, which is independent of redox condition; however, the morphology of the insoluble aggregates is strongly influenced by the environment in which scFv is aggregated.