Objective Myocardial histopathological detection is the gold standard for the diagnosis of myocardial fibrosis, and special staining is a commonly used detection method in pathological examination.However, at present, there are some problems in the software analysis of commonly used special staining methods for myocardial fibers, such as the difficulty in identifying the location of collagen and the large error in quantitative analysis, and it is of great clinical significance to explore the best special staining method for the diagnosis of myocardial fibrosis. Methods Paraffin sections of cardiac tissue were prepared using a transgenic mouse model of cardiomyopathy with specific cTnT R141W gene mutation caused by myocardial tissue mutation constructed in the laboratory, and four staining methods were performed, includind Masson's trichrome staining (Masson), Picric Sirius red staining (PSR), van Gieson staining (VG), and Sirius red/Fast Green staining(SR/FG), and comparative observation. Subsequently, Image J software was used to quantitatively compare the area of collagen fibers.The Sirius red/Fast green staining method was optimized from three aspects: dye concentration, staining time, and acid solution prestaining, and the subsequent quantitative analysis of collagen fibers was applied and verified. Results The distribution of collagen fibers could be observed by the four special staining methods, among which the Sirius red/Fast green staining method, 0.1% Sirius red-picric acid acidic solution was used to pre-stain for 5 min, and the concentration of the dye solution was adjusted to 0.1% Sirius red-picric acid and 0.04% fast green mixture, and the sections were placed in the mixed staining solution for 1h, has the lowest missed reading and loss in determine the proportion of collagen fibers. Conclusion Compared with other traditional collagen fiber special staining methods, the optimized Sirius red/Fast green staining method in this paper has bright coloring between collagen fibers and myocardial tissue, obvious color contrast, good stability, convenience and speed, and is more suitable for subsequent quantitative analysis and application of collagen fiber proportion.