This study presents a simple, inexpensive and environment-friendly fabrication strategy for microfluidic paperbased analytical devices which can resist the penetration of surfactant solutions and organic solvents, by using water-based polyurethane acrylate via UV light curing. The filter paper's barrier created using cured PUA could withstand surfactant solutions (10 wt%, CTAB, SDS and Triton X-100) and organic solvents (methanol, isopropanol, DMF, DMSO, etc). This is very useful for analyzing complicated biological samples on the microfluidic paper-based analytical devices. In addition, the expense of water-based polyurethane acrylate is very cheap (about $8/500 g) and PUA developer is water that is environmental-friendly. To further verify its advantage, we successfully demonstrated the proposed microfluidic devices for detection of E. coli targets in tap water and seawater via colorimetric analysis in a fast and convenient manner. Our results revealed that the linear response to E. coli BL21 was in the range of 10 4 ∼10 9 cfu/mL. The proposed method can effectively avoid the damage for the hydrophobic barriers from the solution even some aggressive liquids, and shows great potential in on-site analysis, environmental monitoring, and food safety.