Stripe rust is one of the most common diseases challenging the safe production of wheat. Rapid identification and analysis of urediospores, responsible for disease transmission, are the key to preventing and controlling stripe rust. The current spore detection is time-consuming and requires auxiliary equipment, but many optical detection methods and sensors with high efficiency, sensitivity, and detection ability have been developed and widely used. Thus, the investigation of optical parameters is the prerequisite for optical sensitive detection and is detailed in this study. Firstly, the microscopic images and surface elements of wheat stripe rust urediospores were obtained using a high-resolution electron microscope and an energy spectrum analyzer. The reflectivity and absorptivity spectra in the near-infrared band of active/inactivate urediospores were measured. Further, the optical parameters, such as reflection phase shift, complex refractive index, and dielectric constant, of the urediospores were analyzed based on electromagnetic theory and Kramers–Kronig relation. The results revealed that the urediospores have a strong tolerance to near-infrared light, and the real part of the complex refractive index n was between 1.0 and 1.4. These observations indicate that whether the spores are active or not has little effect on the real part and dielectric constant of the complex refractive index but has a significant impact on the imaginary part and extinction coefficient of the complex refractive index, and the corresponding relationship between the optical properties of urediospores and biological components was established. These findings were verified with HPLC-MS and Near-Infrared Spectroscopy Analysis Technology and lay a theoretical foundation for detecting urediospores of wheat stripe rust by using optical sensors. The study provides a reference for the analysis of optical characteristics of other biological samples.