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Microdroplet approaches for digital detection of DNA/RNA have been reported in the past years [1-4].However,no droplet based digital detection method has been reported for protein yet,largely due to the lack of a target amplification method for protein.One key step towards single protein molecule detection in microfluidic droplets is the realization of single enzyme molecule detection in emulsion droplets.Herein we report a highly parallel microfluidic droplet method for single enzyme molecule detection.An integrated microfluidic chip was designed for high throughput uniform droplet generation,monolayer droplet collection,incubation,detection and release.As illustrated in Fig.1,the chip is composed of a glass slide,a bottom flat PDMS layer,a patterned layer with a capture well excised off and a cover flat layer.The final integrated chip contains two aqueous inlets,two oil inlets,a droplet generation section,a droplet capture well and an outlet.β-Galatosidase (β-Gal) molecules and fluorogenic substrate fluorescein di-β-D-galactopyranoside (FDG) were injected from two separated inlets to form uniform 20 μm droplets (Fig.2) at the frequency of 6.6 KHz,which were then trapped as monolayer in the capture well (Fig.3A).A series concentration of β-Gal solutions was prepared to generate droplets at single molecule level and analyzed under fluorescence microscope.Ratio of fluorescent droplets increased as the β-Gal concentration increased (Fig.3B-D).Fluorescence intensity distribution of droplets with 0.1,0.5 and 1 enzyme molecules was found to match with Poisson distribution well (Fig 4),proving the feasibility of detecting single enzyme in emulsion droplets.Based on the digital counting method,measured concentrations of the enzyme were correlated linearly to the dilution factors,establishing the reliability and reproducibility of the digital detection method for absolute quantification of enzyme molecules (Fig.5).By integrating with single molecule ELISA in droplets,a general single protein molecule detection method could be easily achieved.