Synthesis and targeted delivery of dendrimer-based fluorescent biosensors in living HeLa cells are reported. Following electroporation dendrimers are shown to display specific subcellular localization depending on their size and surface charge and this property is preserved when they are functionalized with sensing moieties. We analyze the case of double dendrimer conjugation with pH-sensitive and pH-insensitive molecules leading to the realization of ratiometric pH sensors that are calibrated in vitro and in living cells. By tuning the physicochemical properties of the dendrimer scaffold sensors can be targeted to specific cellular compartments allowing selective pH measurements in different organelles in living cells. In order to demonstrate the modularity of this approach we present three different pH sensors with tuned H+ affinity by appropriately choosing the pH-sensitive dye. We argue that the present methodology represents a general approach toward the realization of targetable ratiometric sensors suitable to monitor biologically relevant ions or molecules in living cells.