The biggest challenge in current isolation methods for lipid bilayer‐encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large‐scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near‐native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size‐based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost‐effectiveness and rapid enrichment of the vesicles align with demands for large‐scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery.