Proteins are essential components of organisms and are involved in a wide range of biological functions.Protein is also the biomarker of many diseases.However,in the early stages of disease,these protein biomarkers are present at ultra-low levels.Thus,the ability to detect ultra-low concentrations or even single protein molecules in serum directly could accelerate the discovery and use of more sensitive diagnostic biomarkers.Micro fluidics allows the manipulation of small quantities of reagents in a high-throughput manner and is therefore highly amenable to single protein characterization and more generally to digital analysis.Here we proposed a high-throughput droplet based single-molecule ELISA system capable of detecting the presence of single proteins in emulsion droplets thus allowing highly accurate and sensitive digital detection of proteins.In the work,we captured the proteins on microscopic beads decorated with specific antibodies and then labeled the immuno-complexes(one or zero labeled target protein molecules per bead)with an enzymatic reporter capable of generating a fluorescent product.After diluting and randomly dispersing the magnetic beads into microdroplets and ensuring that each droplet contains only one bead at most,we used fluorescence imaging to detect single protein molecules.Our microfluidic droplet-based single-molecule enzyme-linked immunosorbent assay(digital ELISA)approach allows detection of clinically relevant proteins in with higher sensitivity than conventional ELISA.We anticipate this digital ELISA system will achieve utlrahigh sensitive detection of proteins,thus discover and validate new disease associations and define a new era of molecular diagnostics.