The measurement of intracellular analytes has been key in understanding cellular processes and function, and the use of biological nanosensors has revealed the spatial and temporal variation in their concentrations. In particular, ratiometric nanosensors allow quantitative measurements of analyte concentrations. The present review focuses on the recent advances in ratiometric intracellular biological nanosensors, with an emphasis on their utility in measuring analytes that are important in cell function.