It is reported that Cathepsin L (CatL) activity is correlated with actinic keratosis and squamous cell cancers. The purpose of this study is to propose an on-site testing of CatL activity in the skin, which can be used for the diagnosis of skin conditions noninvasively, easily, quickly and at any time. In this study, a novel immunochromatographic test-strip is proposed for CatL analysis in the skin. In order to realize the disposable test-strip, a colloidal gold technique was selected as the molecular recognition method for the CatL. A human skin sample was collected using tape stripping. The fabricated immuno-chromatographic test-strip (test-strip) consists of four pads, a sample pad, a conjugate release pad, a nitrocellulose membrane and an absorption pad. Based on optimal assay conditions, the sensitivity of the test-strip was measured. It required 90 s for a sample to reach the test-line on the test-strip. The optical density was proportion to the activity of CatL. In order to cancel out the influence of the collecting technique, the CatL/TP ratio was evaluated. It was suggested that the test-strip might be used for the qualitative analysis of CatL in the stratum corneum.