Single-molecule techniques have been used successfully to visualize real-time enzymatic activities, which reveals transient complex properties and heterogeneity of various biological events. Especially, conventional force spectroscopy including optical tweezers and magnetic tweezers has been widely used to monitor the change in the DNA length by enzymes with high spatiotemporal resolutions of ~nanometers and ~milliseconds. However, DNA metabolism results from the coordination of a number of components during the processes, which requires to efficiently monitor the complex of proteins catalyzing DNA substrates. In this min-review, we will introduce a simple and multiplexed single-molecule assay to detect DNA substrates catalyzed by enzymes with high-throughput data collection. We conclude with a perspective of the possible directions that enhance the capability of the assay to reveal complex biological events with better resolution.