As an excellent alternative, the vaginal route has been widely investigated to deliver different types of drugs. However, the delivery of hydrophobic drugs via conventional preparation is challenging. Here, for the first time, we investigated using PEG as a permeation enhancer in thermosensitive mucoadhesive hydrogels containing Nile red as a model of the hydrophobic compound. Pluronic® F127 (P127) and Pluronic® F68 (P68) were used as thermosensitive agents, and HPMC was used as mucoadhesive agents. The results showed that the concentration of PEG did not change the gelation temperature, mucoadhesive properties, pH, recovery, and rheological behavior of hydrogels. Finally, using PEG could improve the ex vivo permeation and retention profiles of Nile red up to 20-folds and 15-folds, respectively. Following the promising results of this proof of concept study, the application of PEG in improving the vaginal delivery of therapeutic agents should be carried out.