Enterovirus 71 (EV71) is the main causing agent of hand-foot-mouth disease (HFMD), however, the specific antiviral agents are still not commercially available. In order to find antiviral agents against EV71, a high throughput drug-screening model targeting EV71 3C^pro was established and a small-molecular compound library was screened. The virus EV71-MZ was isolated from an HFMD patient, and identified by PCR. A 3C^pro recognition site was inserted into the middle region of YFP open reading frame to generate the mYFP by insertion mutation. The full length mYFP proteins were observed by fluorescence microscope and the protein level was measured by using microplate reader in Ex(500 nm)/Em(535 nm). Change of the fluorescence value reflected the degree of the inhibition on 3C^pro activity. A small-molecular compound was screened by using the established screening model in the high throughput drug screening system, then the antiviral activity of the active compounds was further evaluated by plaque assay. As a result, mYFP expressed well in 293A cell; the expression of 3C^pro reduced the fluorescence signal remarkably, however, the signal was recovered by adding Rupintrivir, an inhibitor of 3C^pro. These results indicated that the screening model targeting 3C^pro was established successfully. 26 of 26 000 compounds significantly reverted the fluorescence signal of the mYFP in the presence of 3C^pro; noticeably, two of the 26 compounds, i.e. numbering 3 and 8, exhibited strong antiviral activity by plaque assay. All together, 3C-mYFP co-expression system is an optimized and effective screening method for high-throughput screening of anti-EV71 3C^{pro drugs.}