Objective To establish a multiple special staining method combined with image processing protocols tailored for bone tissue with varying degrees of decalcification. Methods Fifteen 10-week-old SpragueDawley rats were selected and humanely euthanized. The right femurs were harvested and fixed, followed by acid decalcification at different intensities. Paraffin-embedded sections were prepared and stained with hematoxylin-eosin to evaluate the degree of decalcification. The sections were then stained with toluidine blue, safranin O-fast green, and a multiple special staining protocol, and the staining efficacies of each method in terms of bone morphology under varying decalcification conditions were compared. Spectral unmixing techniques were also employed to develop an image processing strategy specifically for multiple-stained samples. Results Toluidine blue and safranin O-fast green staining showed suboptimal result in over-decalcified samples, while the optimized multiple special staining protocol provided enhanced visualization of cortical bone, chondrocytes, and collagen structures in over-decalcified tissues.Spectral processing resolved composite images into individual dye-specific components, thereby improving the accuracy of the quantitative analysis. Conclusions The established multiple special staining method significantly improves the visualization of cortical bone, cartilage, and collagen in over-decalcified samples. This technique offers a valuable tool for morphological studies related to bone metabolic diseases.