The invention discloses a pepsin detection kit and a detection method. The kit comprises a test strip and reaction liquid, wherein the test strip comprises a body which comprises a backing bottom plate; a sample binding pad, a nitrocellulose membrane and a water absorption membrane are arranged on the backing bottom plate; the sample binding pad and the water absorption membrane are respectively overlaid at two ends of the nitrocellulose membrane; a detection line is arranged on the nitrocellulose membrane, and is coated with a monoclonal antibody for resisting pepsin substrate; and the reaction liquid contains marked pepsin substrate. According to the pepsin detection kit, by utilizing the characteristic that the reactionogenicity of substrate is lost due to the protein digestion characteristic of pepsin, pepsin is indirectly qualitatively and/or quantitatively detected by detecting the quantity of the substrate with reactionogenicity remained after being digested by pepsin, so that the pepsin detection kit has the characteristics of good sensitivity, high precision and easy operation.