Immunotherapy has become an efficient treatment method of breast cancer. Detection of proteins such as PD-L1 and CTLA-4, which are important immune checkpoint molecules, is attracting more and more attention as they play key roles in immunotherapy. Here, by combining the high resolution of DNA-PAINT (DNA points accumulation for imaging in nanoscale topography) with the qPAINT quantitative analysis method, accurate spatial localization and absolute quantification of PD-L1 and CTLA-4 on the membrane of breast cancer cells could be achieved. Meanwhile, exchange-PAINT was also conducted to count three other biomarkers (EpCAM, EGFR, and HER2). Simultaneous analysis of these biomarkers can greatly facilitate the differentiation of different kinds of breast cancer. Such a simple quantitative analysis method holds great potential in diagnosis and immunotherapy of cancers.