The precise and efficient sorting of microscopic particles is critical in diverse fields, including biomedical diagnostics, drug development, and environmental monitoring. Fluorescence imaging-activated sorting refers to a strategy where fluorescence images are used to dynamically identify target particles and trigger selective manipulation for sorting purposes. In this study, we introduce a novel microfluidic particle sorting platform that combines optical tweezers with real-time fluorescence imaging for detection. High-speed image analysis enables accurate particle identification and classification, while the optical trap is selectively activated to redirect target particles. To validate the system’s performance, we used 10 µm green and orange fluorescent polystyrene particles. The platform achieved a sorting purity of 94.4% for orange particles under continuous flow conditions. The proposed platform provides an image-based sorting solution, advancing the development of microfluidic systems for high-resolution particle sorting in complex biological and environmental applications.