In order to enhance the added value of Tetrodontiformes fasciatus and investigate the effects of different preparations on the extracted collagen from skin of T. fasciatus, collagens extracted from the skin of T. fasciatus by acid, hot water, pepsin, papain, and ficin were used to evaluate chemical and structural properties, respectively. In this study, characterization of collagen from T. fasciatus skin in term of the extraction yield, amino acid composition, Fourier transform infrared (FTIR) spectroscopy, ultraviolet spectroscopy(UV), scanning electron microscopy(SEM) and sodium dodecyl sulfate-polyacrylamide gel electropheresi (SDS-PAGE) was studied. Results showed that the extraction yield of collagen obtained by pepsin was the highest, the amino acid compositions were similar and the contents were different; the content of proline in collagen extracted by 3 enzymes is significantly lower than that of collagen extracted by acid and hot water. FTIR showed collagen obtained by the five extraction methods had Amide A, Amide B, Amide I, Amide II and III, and confirmed the triple helical structure of the collagens. UV showed a strong absorption peak around 235 nm, indicating a typical collagen when combined with FTIR. SDS -PAGE analysis confirmed that collagen in skin of T. fasciatus was type I collagen, and preparations by acid and pepsin retained β, α1 and α2 chains of collagen, while papain disassociate collagen and obtained protein with low molecular mass; SEM showed that extracted collagen by acid was most suitable for biomedical materials. Therefore, different extraction methods resulted in different chemical and structural properties of fish skin collagens. The molecular weight distribution of collagen extracted by different enzymes had obvious differences. According to research needs, different preparations can be selected to develop collagen products.