Abstract In order to confirm that the cholesterol end groups of cholesterol-end capped polyethylene glycol really insert into the liposome bilayer and investigate how the incorporation affects the microenvironment of liposome bilayer, two kinds of molecular probes, namely Nile Red and pinacyanol chloride, were used. Their UV–visible and fluorescence spectrum were recorded before and after the addition of the polymer. Shifts of the maximum absorbance (λmax) of Nile Red show that the bilayer microenvironment around Nile Red is becoming more polar with increasing polymer concentration while shifts of λmax of pinacyanol chloride indicate that the surrounding environment of pinacyanol chloride is becoming more apolar with addition of polymer. Effect of composition of liposome was also studied. With high ratio of dimethyldioctadecylammonium bromide (DODAB) fraction in liposome, λmax of Nile Red is more easily affected by the addition of Chol-PEG-Chol while liposome with cholesterol shows relatively high stability to the addition of Chol-PEG-Chol.