Plastics, recognized for their convenience, disposability, and recyclability, have emerged as a significant ecological challenge, particularly with the prevalence of microplastics (MPs, 1 μm – 5 mm) and sub-micron MPs (100 – 1000 nm) in natural environments. While extensive research has focused on their occurrence and environmental impacts, quantification methods developed for MPs exhibit limitations when applied to sub-micron MPs due to their smaller size. This study addresses these limitations by introducing a novel monitoring system that integrates fluorescence labeling with a microfluidic device and particle tracking software, enabling automated quantification and size measurement of both spherical and fragmented MPs of size in the sub-micrometer range. Results showed that the developed system enabled fast quantification and size measurement of 500- and 1000-nm polystyrene (PS) sub-micron MP beads and fragmented PS and polyvinyl chloride (PVC) sub-micron MPs. Additionally, fluorescence labeling enabled the real-time discrimination of PS and PVC sub-micron MPs. Lastly, the microfluidic system allowed the monitoring of sub-micron MPs within a small quantity of water samples. This automated system has a high potential for swift and real-time monitoring of sub-micron MPs in the environment. By enhancing our ability to detect and quantify sub-micron MPs, this study contributes to a more comprehensive understanding of their presence and distribution in environmental systems.