2D Gel Electrophoresis (2DGE) image is the most widely used method for the isolation of the objective protein by comparative analysis of the protein spot pattern in the gel plane. The process of protein analysis is the method, which compares the protein pattern that is spread in the gel plane with the contrast group and finds interesting protein spot by image analysis. Previous 2DGE image analysis is composed of gaussian fitting, and segments protein spots by watersheds, a morphological segmentation. Watersheds have a benefit that is fast in global threshold, but induces under-segmentation and over-segmentation of spot area when gray level is continuous. The drawback was somewhat solved by marker point institution, but needs the split and merge process. This paper introduces a novel segmentation of protein spots by watersheds using hierarchical threshold, which can resolve the problem of marker-driven watersheds.