To further increase the mechanical properties of polyacrylonitrile-based carbon fibers, a multiple stretching technique was applied. Carbon fibers were multiple stretched at 2200 °C and characterizations such as SEM, Raman, XRD, and TEM were used to investigate the evolution of microstructure of carbon fibers. It was found that the grooves on the surface of carbon fibers along the fiber axis direction became more obvious and the cross-section of fibers were twisted from nearly circular to elliptical after multiple stretching. Growth and slippage of graphite microcrystals along the fiber axis direction resulted decrease in disordered structure and defects in the carbon fibers and increase in the degree of graphitization. The multiple stretching effectively enhanced the length-to-width ratio of microcrystals. An increase of 75 GPa in tensile modulus and a retention rate of 0.95 in tensile strength were realized for carbon fibers multiple stretched at 2200 °C.