Single-cell analyses of secretory proteins are essential to fully understand cellular functional heterogeneity and unravel the underlying mechanisms of intercellular signaling and interactions. Retrieving dynamic information of protein secretion at single-cell resolution reflects the precise, real-time functional states of individual cells in physiological processes. Such measurements remain very challenging in single-cell analysis, which requires highly integrated systems capable of performing on-chip single-cell isolation and subsequent real-time protein detection. Here, recent advances in microfluidics-based single-cell manipulation and emerging approaches for label-free single-cell biosensing are reviewed. The advantages and limitations of these technologies are summarized and challenges to establish the integrated microfluidic biosensing systems for real-time single-cell secretomics are discussed. Recent efforts on integrated platforms for on-chip single-cell protein assays are highlighted and some perspectives on future directions in this field are provided.