Objective The existing dyeing Methods of myocardial fibrosis were optimized to make up for the problems of missing and misreading of collagen fibers in the quantitative analysis of the current common dyeing Methods of myocardial fibers, and to provide a reference for the semi-quantitative and diagnosis of myocardial fibrosis. Methods Paraffin sections of cardiac tissue were prepared using a transgenic mouse model of cardiomyopathy with a specific laboratory-constructed cTnT R141W gene mutation. Four staining method were performed for comparative observations:Masson’s trichrome(Masson) staining, picrosirius red( PSR) staining, van Gieson(VG) staining, and Sirius red / fast green(SR/ FG) staining. Image J 2. 1. 0 software was used to quantitatively compare the areas of collagen fibers. SR/ FG was optimized from three aspects: dye concentration, staining time, and acid solution prestaining, and the quantitative analysis of collagen fibers was then verified. Results The collagen fiber distribution was observed by the four staining method, among which SR/ FG was notable. It involved prestaining with a 0. 1% Sirius red-picric acid acidic solution for 5 min, adjusting the concentration of the dye solution to 0. 1% Sirius red-picric acid and 0. 04% fast green mixture, and incubating the sections in the mixed staining solution for 1 h. This method exhibited the lowest incidence of missed readings and loss in determining the proportion of collagen fibers. Conclusions Compared with other traditional collagen fiber staining method, the optimized SR/ FG technique described in this paper produces bright coloring of collagen fibers and myocardial tissue, obvious color contrast, and high stability, convenience, and speed. It is suitable for subsequent quantitative analysis and determination of the collagen fiber proportion.