Proteolytic degradation of the extracellular matrix represents a key step in cancer dissemination and metastasis. To probe cellular proteolytic activity, fluorescent sensing substrate was developed, yet prior studies focused on average activity of thousands of cells. Considerable evidence suggests a specialized subset of cancer cells are driving metastasis, highlighting the value of single-cell approach to reveal cancer cellular heterogeneity. In addition, when only a small number of cells are available, single-cell analysis is required to draw a statistical conclusion. Here, we present a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small number (10–100) of cells can be individually characterized. Furthermore, the platform allows monitoring single cells at multiple time points for the investigation of dynamics in proteolytic activity. The presented platform represents a simple and reliable tool for single-cell proteolytic analysis, illuminating the heterogeneous and dynamic nature of cancer cells.