Droplet microfluidics is a powerful platform for high-throughput single-molecule protein analysis. However, the issues of coalescence and crosstalk of droplets compromise the accuracy of detection and hinders its wide application. To address these limitations, a novel colloidosome-based method was presented by combining a Pickering emulsion with droplet microfluidics for single-molecule protein analysis. Utilizing the self-assembly of easily synthesized colloidal surfactant F-SiO2 NPs at the water/oil interface, the colloidosomes are rigidly stabilized and can effectively avoid the leakage of fluorescent molecules. The crosstalk-free colloidosomes enable high-throughput single-molecule protein analysis, including heterogenous dynamic studies and digital detection. As a robust and accurate method, colloidosome-based microfluidics is promising as a powerful tool for a wide variety of applications, such as directed enzyme evolution, digital enzyme-linked immunosorbent assay (ELISA), and screening of antibiotics.