Objective For tissues that require continuous diastole and systole in vivo, such as the heart, there are many problems with traditional tissue fixation method. This study verified and evaluated selection of a potassium chloride termination concentration and procedure from the animal heart sampling process and compared it with traditional and active perfusion fixation method to optimize sampling and fixation method of heart tissue in small experimental animals such as rats. Methods Intracardiac injection of a KCl solution in situ and transposition of a KCl solution after heart extraction were compared and analyzed. Then, the reduction degree of heart morphology in vivo, the consistency of diastolic termination, the success rate of modeling, the morphology of the myocardium, and the application of specialized staining were analyzed. The traditional fixation method, active perfusion fixation method, and KCl arrest were systematically compared. Results In the traditional passive fixation method, the overall shape of the heart was irregular, and some samples showed ventricular collapse, obvious contraction of the epicardium, and obvious thrombus in the ventricular lumen and myocardial vessels. Because of the inconsistency in the arrest period, heart morphology varied greatly among individuals. In the active perfusion method, the overall size of the heart was significantly increased, the double ventricle was severely dilated, muscle fibers were broken, and the microstructure was damaged. In KCl arrest, the overall heart shape was intact, there was no obvious contraction of the epicardium, the arrest period was relatively consistent, and there was little difference between individuals. Conclusions Modified KCl arrest is superior to traditional passive fixation method and active perfusion method in terms of the in vivo shape reduction of the heart and subsequent analysis by histopathological staining. This method provides a reference for pathological and histological studies of cardiac tissue fixation method ology. Furthermore, the difference between individual heart tissue morphological analysis of the consistency and in vivo shape reduction, the animal model of the heart internal fine structure, such as left and right ventricles, left and right atria, and the ventricular septum, papillary muscle, valves, such as analysis of observation, is of great significance.