In vivo cell tracking can provide information on cell migration and accumulation in the organs. Here, both folate and amino modified polymer dots were synthesized and screened for in vitro and in vivo tracking of macrophage Ana-1 cells. Flow cytometry analysis demonstrated that prepared polymer dots showed cellular uptake of approximately 98% within a short incubation time of 2 h, and these polymer dots maintained a cell labeling rate over 97% after 2 d. Moreover, a CCK-8 assay suggested that these polymer dots increased Ana-1 cell viabilities up to 110% at concentrations from 5 to 50 μg mL⁻¹. Furthermore, the in vivo real time imaging of labelled Ana-1 cells in the alveolus of lung and lymph nodes were clearly detected by probe-based confocal laser endomicroscopy (pCLE). This study demonstrates a unique approach using polymer dots for real-time high resolution tracking of macrophage cells in deep organs and the lymphatic system.