The invention discloses construction of a portable glucometer-based paper analysis device for thrombin detection. A hydrophobic wax printing pattern of a microfluidic paper chip is designed by an Adobe illustrator CS4 software on a computer; gold nanoparticles grow in a hydrophilic working area of the paper chip, a hairpin-structured probe containing a thrombin aptamer chain is loaded, and a target object is captured; and a sucrose-labeled recognition chain and the hairpin structure opened probe are hybridized, glucose is generated by the catalytic hydrolysis effect of sucrose on cane sugar so as to further realize detection of thrombin through a portable glucometer.