Cells infiltrating lesional skin cannot be analyzed in detail through immunohistochemistry or cell separation techniques using skin biopsy specimens. Here, we describe a skin liquid biopsy method that enables T cell isolation from small amounts of lesional whole blood from mycosis fungoides (MF) patients. Gene expression analysis was performed on isolated cells which revealed that CD4+CD45RO+ T cells contribute to the pathogenesis of MF, and CD8+CD45RO+T cells serve as the effector cells of CD4+ malignant MF. CD8+CD45RO+ T cells in lesional blood negatively correlate with the modified severity-weighted assessment tool (mSWAT) and negatively regulate cellular responses. CD4+CD45RO+ T cells highly express TNC, C1QB, PLK4, SGK1, RGS1 and CD69, and have a lower diversity of the T-cell receptor repertoire. Our results provide new insights into the pathogenesis of MF. The technique described here may be applicable to the evaluation of treatment efficacy for other skin inflammatory diseases.