In the field of asphalt material science, accurate observation and analysis of the microstructure of polymer-modified asphalt are crucial for understanding its performance and optimizing production processes. This study systematically investigates the sample preparation methods for fluorescent microscopy of polymer-modified asphalt. By analyzing the impact of different preparation methods on the observation of the micromorphological structure of modified asphalt, this study compares the hot-drop asphalt slide formation method and the frozen asphalt formation method with section extraction. The research found that covering with a cover slip can improve image clarity, but heating the bottom of the slide can change the phase state of asphalt. Based on the experimental results, the slide-cover method of sample preparation is recommended, which is simple to operate, has clear imaging, and good repeatability in observation. The final sample preparation process includes pouring heated modified asphalt onto a slide, covering with a cover slip by free-falling method, and observing after cooling at room temperature. This study not only provides a standardized sample preparation method for the microstructural analysis of modified asphalt but also highlights the potential of fluorescence microscopy in interdisciplinary applications, especially in the collaborative research of material science, chemical engineering, and transportation engineering. It offers important technical support for the performance evaluation and application of modified asphalt.