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 hinder 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.