One of the critical prerequisites for accurately measuring the mechanical properties of profiled fibers is the precise determination of their cross-sectional areas (CSAs). In this study, a new method is established for determining a single profiled fibers’ CSA based on the frozen section method and digital photo, pixel-ratio method (FS-DP). FS-DP is used to obtain a transverse section of a fiber, by acquiring an image of the cross section using optical microscopy or scanning electron microscopy, and then calculating the CSA using Photoshop. Using FS-DP, it was found that the shape of a fiber of silk changes little in a range of 50 μm, but varies considerably over a range of 1 m, while the CSA of cocoon silk (900 m) first increases and then decreases. Mechanical property tests showed that the elongation, strength, elastic modulus, and toughness values of the cocoon silk are consistent with those reported previously. Additionally, FS-DP was also used to observe other profiled fibers. The application tests indicated that FS-DP can be used to quickly and accurately obtain the CSA of a single profiled fiber, and that it is suitable for the large-scale determination and analysis of the mechanical properties of profiled fibers.