In this study we show that steady-state fluorescence anisotropy within PEBBLEs (Probes Encapsulated By Biologically Localized Embedding) can be used for the optochemical sensing of analytes such as Zn2+, O2, and Ca2+. Steady-state fluorescence anisotropy is a non-time resolved method that measures a combination of rotational and fluorescence lifetimes. This eliminates the need for reference dyes and ratiometric techniques to obtain quantitative results, even when using intensity-based sensor dyes. An advantage to working with PEBBLE nanosensors is that the encapsulated dye is localized in a constant rotational environment. This is in contrast to the use of free dyes, which can be affected by interferents such as protein binding.