Silkworm silk has excellent mechanical properties, biocompatibility, and promising applications in the biomedical sector. Silkworms spin silk at the beginning and end of each of their five instar stages, as well as spinning mature silk after the fifth instar. We evaluated the mechanical properties and structure of 10 kinds of silk fibers from different stages. A tensile test showed that instar beginning silk, instar end silk, and mature silk possess distinct properties. Attenuated total reflectance Fourier-transform infrared spectroscopy and X-ray diffraction results showed that the excellent mechanical properties of instar end silk are attributed to higher beta-sheet content and suitable crystallinity. Liquid chromatography tandem mass spectrometry showed that P25 protein content in IV-E silk is 2.9x higher than that of cocoon silk. This study can offer guidelines for further biomimetic investigations into the design and manufacture of artificial silk protein fibers with novel function.