The identification of T and B lymphocytes has relied on using antibodies against different biomarkers as the gold standard. Emerging small molecule-based fluorescent probes have the potential to replace antibodies. Herein, we report the first human-T-cell-selective fluorescent probe, Mito thermo yellow (MTY), achieving the live T cells' distinction from B cells, which was previously impossible without the help of antibodies. The unexpected cell selectivity of MTY is attributed to the higher mitochondria mass and membrane potential of T cells over B cells. This study enriches the toolbox for live cell distinction from complex cell communities.