The invention relates to the technical field of medical detection, particularly to a urine detection test paper for reflecting the human fatigue degree, and a preparation method thereof, wherein the urine detection test paper contains thiol protease, sodium dihydrogen phosphate, water-soluble polythiophene, chloroauric acid tetrahydrate, sodium hydroxide and polyvinyl alcohol, and the surface of the urine detection test paper is coated with a chitosan/titanium dioxide composite film. The preparation method comprises: immersing filtration paper in the mixed solution of a solution A and a solution B, and covering the surface of the filtration paper with a chitosan/titanium dioxide composite film, wherein the solution A contains water-soluble polythiophene, chloroauric acid tetrahydrate, sodium dihydrogen phosphate and sodium hydroxide, and the solution B contains polyvinyl alcohol and thiol protease. According to the present invention, the prepared urine detection test paper has advantages of strong hydrophilicity, convenient operation and high detection sensitivity, and can adsorb and permeate urine protein.