In this study, aqueous nanodroplets with different concentrations of the dye Trypan Blue are synthesized and investigated within W/O microemulsions. The AOT surfactant is used to stabilize the resulting nanodroplets in n-hexane solvent. The molar ratio of water to surfactant is kept constant to evaluate the role of Trypan Blue concentration on the photophysical and structural properties of the microemulsions using various characterization techniques. Additionally, these systems are studied at different mass fractions of droplets (MFD). DLS data indicate that the hydrodynamic diameters of the nanodroplets range between 109 and 169 nm, while also showing that the droplet size decreases with increasing Trypan Blue content. Furthermore, PDI values range from 0.138 to 0.201, indicating moderate polydispersity of the synthesized particles. The UV-Vis spectra show a hypsochromic shift in λmax from 562 to 594 nm, corresponding to a decrease in polarity within the confined droplet medium. Moreover, the absorption intensity is found to increase for higher MFD values. The fluorescence spectroscopic results reveal that increasing MFD is accompanied by an enhancement in emission intensity, whereas no significant change occurs in the emission wavelength. The calculations show that ET(30) values range from 48.1 to 50.9 kcal/mol, indicating a lower polarity of the droplet core compared to bulk water. Overall, the findings suggest that the photophysical properties of these microemulsions can be optimized and controlled, with desirable stability observed at moderate dye concentrations and MFD levels.