The comprehensive characterization of novel dextran nanoparticles with regard to their suitability as pH-sensors for analytical applications (e.g. in physiology) is described. The nanoparticles are labeled with both a pH-indicator dye (fluorescein isothiocyanate, FITC) and a reference dye (sulforhodamine B acid chloride) as an internal standard. The fluorescence intensity of FITC increases with increasing pH, whereas the signal of the reference dye remains constant. Plotting the ratio of both signals against the pH gives a pK(a) of 6.45, which is appropriate for most of the measurement purposes. Furthermore, the influence of temperature, ionic strength and oxidizing substances on the performance of the fluorophores inside the dextran nanoparticles is examined. These results are compared to the dissolved dyes in order to evaluate if the dextran matrix affects the fluorescence properties of the sensor and the reference dye, and whether or not these nanosensors are suitable for pH-sensing in biological samples.