Ionophores are widely used ion carriers in ion selective sensors. The effective stability constant (β) is a key physical parameter providing valuable guidelines to the design of ionophores and carrier-based ion selective sensors. The β value of ion-carrier complex in plasticized poly(vinyl chloride) (PVC) membranes and solutions have been determined in the past by various techniques, but most of them are difficult to implement at the nanoscale owing to the ultrasmall sample volume. A new methodology based on charged solvatochromic dyes is introduced here for the first time to determine β values directly within ion selective nanospheres. Four ionophores with different selectivities toward Na+, K+, Ca2+, and H+, respectively, are successfully characterized in nanospheres composed of triblock copolymer Pluronic F-127 and bis(2-ethylhexyl) sebacate. The values determined in the nanospheres are smaller compared with those in plasticized PVC membranes, indicating a more polar nanosphere microenvironment and possible uneven distribution of the sensing components in the interfacial region.