The exploration of cytology mechanisms of nanosilver uptake, toxicity, and detoxification has become an important issue due to its widespread applications. Previous studies have shown differences in the toxic response of mammalian cells to nanosilver. However, the analysis results based on cell populations ignore the impact of cell uptake heterogeneity on the expression of associated stress proteins and cellular physiological activities. In this respect, this work investigated the interaction between silver uptake and metallothionein (MT) expression in individual cells. In addition, we have also preliminarily elucidated the sensitivity variation to AgNPs by using five cell lines, e.g., LX-2, HepG-2, SK-HEP-1, Huh-7, and MDA-MB-231, by adopting a two-dimensional (2D) high-throughput single-cell analysis platform coupling laser-induced fluorescence (LIF) and inductively coupled plasma mass spectrometry (ICP-MS). We developed a 2D data analysis method for one-to-one unification of fluorescence-mass spectrometry signals corresponding to a specific single cell. It indicated that there is no obvious correlation between cellular silver uptake and cell size, and the low MT expression of cells is more sensitive to silver nanoparticles. For each cell line, significant heterogeneity in MT expression was observed. This provides important information for understanding the potential heterogeneous effects of nanosilver on mammalian biological systems. Overall, detoxified cells are more tolerant to nanosilver and normal cells are more tolerant than cancer cells.