Thymic stromal lymphopoietin (TSLP) is known for its capacity to induce CD11c+ myeloid dendritic cells to promote T helper type 2 (Th2)-skewed inflammatory responses. Although increased expression of TSLP was reported in the lesional skin of limited numbers of patients with atopic dermatitis (AD), the relationships between the degree of TSLP expression in the skin and the severity of AD, epidermal barrier function and eruption type remain to be elucidated. The aim of this study was to examine the relationships between the degree of TSLP expression in the skin and the severity of AD, eruption type and epidermal barrier function using a non-invasive method in a sizeable group of the patients. Stratum corneum tissue was obtained from AD patients by tape stripping, and the stratum corneum TSLP (scTSLP) expression level was evaluated using a TSLP-specific antibody followed by image analysis. The correlations between the scTSLP intensity and the severity scoring of AD (SCORAD) index and epidermal barrier function, such as stratum corneum hydration and transepidermal water loss (TEWL), were analysed. The changes in the scTSLP level induced by the application of moisturizer were also examined. The scTSLP expression level was increased in AD patients compared with healthy subjects and was correlated with SCORAD, especially with the dry skin score, and stratum corneum hydration. Moisturizer application resulted in reduced scTSLP levels. The scTSLP level can be used as a biomarker of AD severity and particularly epidermal barrier status.