As a kind of unicellular eukaryotic protozoa, Tetrahymena is located at the bottom of the aquatic food webs and plays an essential role in the bioaccumulation of mercury (Hg). To track Hg in individual Tetrahymena, a capillary single-cell inductively coupled plasma mass spectrometry (ICPMS) online system was developed. The experimental and instrumental conditions were optimized to ensure the signal detected was the Hg uptake in individual Tetrahymena. Moreover, a quantitative method was established and validated by detecting Hg2+ standard solutions. The limit of quantity was calculated to be approximately 3.8 × 10-15 g Hg/cell, and the detection limit for Hg2+ exposure of Tetrahymena was 0.05 μg/L. By using the proposed method, we found the peak became wider with increasing of exposure concentrations, indicating the accumulated Hg by different Tetrahymena varied greatly, and the difference was more significant at higher exposure concentration. This novel method has the advantages of high sensitivity and real-time detection in individual Tetrahymena, and it could be widely used for further tracking the accumulation of mercury and other metals at the single cell level.