There is a great deal of interest in the development of nanoparticles for biomedicine. The question of how many nanoparticles are taken up by cells is important for biomedical applications. Here, we describe a fluorescence method for the quantitative measurement of the cellular uptake of polymer dots (Pdots) and a further estimation of intracellular Pdots photosensitizer for fluorescence imaging and photodynamic therapy. The approach relies on the high brightness, excellent stability, minimal aggregation quenching, and metalloporphyrin doping properties of the Pdots. We correlated the single-cell fluorescence brightness obtained from fluorescence spectrometry, confocal microscopy, and flow cytometry with the number of endocytosed Pdots, which was validated by inductively coupled plasma mass spectrometry. Our results indicated that, on average, ∼1.3 million Pdots were taken up by single cells that were incubated for 4 h with arginine 8-Pdots (40 μg/mL, ∼20 nm diameter). The absolute number of endocytosed Pdots of individual cells could be estimated from confocal microscopy by comparing the single-cell brightness with the average intensity. Furthermore, we investigated the cell viability as a result of an intracellular Pdots photosensitizer, from which the half maximal inhibitory concentration was determined to be ∼7.2 × 105 Pdots per cell under the light dose of 60 J/cm2. This study provides an effective method for quantifying endocytosed Pdots, which can be extended to investigate the cellular uptake of various conjugated polymer carriers in biomedicine.