Analysis of chemical or biomolecular contents in a tiny amount of specimen presents a significant challenge in many biochemical studies and diagnostic applications. In this work, we present a single-layer, optofluidic device for real-time, high-throughput, quantitative analysis of droplet contents. Our device integrates an optical fiber-based, on-chip detection unit with a droplet-based microfluidic unit. It can quantitatively analyze the contents of individual droplets in real-time. It also achieves a detection throughput of 2000 droplets per second, a detection limit of 20 nM, and an excellent reproducibility in its detection results. In a proof-of-concept study, we demonstrate that our device can be used to perform detection of DNA and its mutations by monitoring the fluorescent signal changes of the target DNA/molecular beacon complex in single droplets. Our approach can be immediately extended to a real-time, high-throughput detection of other biomolecules (such as proteins and viruses) in droplets. With its advantages in throughput, functionality, cost, size, and reliability, the droplet-based optofluidic device presented here can be a valuable tool for many medical diagnostic applications.