In this study, we used one-pot A 2 + B 3 polymerizations to synthesize two aliphatic + alicyclic polymer dots (PDs) having non-conjugated hyperbranched structures, employing two types of dianhydrides as the A 2 components, possessing bridged bicyclic alkene (PD-BT) and non-alkene (PD-ET) units, and Jeffamine T403 polyetheramine (T403) as the B 3 components. We prepared PD-ET from commercially available ethylenediaminetetraacetic dianhydride (EDTAD, A 2 ) and T403 (B 3 ) and PD-BT from bicyclo[2.2.2]oct-7-ene-2,3,5,6-tetracarboxylic dianhydride (BCDA, A 2 ) and T403 (B 3 ). These two types of PDs possessed non-conjugated hyperbranched poly(amic acid) structures with terminal amino functional groups. PD-BT and PD-ET exhibited non-conventional fluorescence with emissions at 435 and 438 nm, respectively, and quantum yields of 12.8 and 14.0%, respectively. The fluorescence intensity of PD-ET was influenced by the pH, but PD-BT was less affected because of its rigid aliphatic bridged bicyclic structure. In aqueous solutions, the sizes of the PD-BT and PD-ET nanoparticles were 3−5 nm, and their net charges can be adjusted by varying the pH. These PDs were non-cytotoxic toward human MCF-7 breast cancer cells and human keratinocyte HaCaT cells at concentrations of 50 μg mL −1 for PD-BT and 500 μg mL −1 for PD-ET. Confocal microscopic bioimaging revealed that the PDs were located within the cells after treatment for 6 h. These PDs were easy to prepare, highly water-soluble, and possessed a large number of peripheral functional groups for further modification. Combined with their non-conventional fluorescence, they appear to have potential uses in bioimaging and as druglabeling carriers.