Temperature-sensitive liposomes are drug carriers that can encapsulate drug molecules and carry them safely to their target sites for release. They ensure that the encapsulated drug is released for it to become cellularly internalized at the site of action. They also have a wide range of medical applications, however the most popular is chemotherapy. In this study, the objective was to prepare and purify calcein loaded temperature-sensitive liposomes. Calcien loaded liposomes were prepared using the thin-film hydration method and calcein was loaded passively. To separate non-encapsulated calcein from the loaded liposomes three purification methods were utilized: ion exchange beads, centrifugation, and size exclusion methods. The formulated liposomes were characterized for zeta potential, average particle size, phase transition temperature, and release of loaded calcein molecules at 21, 37, and 45◦ C. Average size of temperature-sensitive liposomes after preparation and purification was < 200 nm and the poly-disparity index < 0.2. The stability and release efficiency of the temperature-sensitive liposome formulation at 37°C in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution and 5% Foetal bovine serum was optimal with a percentage leakage of less than 5% in both media. The release ratio, which was calculated as the ratio of fluorescence intensity of calcein in heated temperature-sensitive liposomes (45°C) to the fluorescence intensity of non-heated temperature-sensitive liposomes at room temperature (R= I21°C/I45°C) was 1.489, the target-release ratio to be achieved was 1.5. The application of temperature-sensitive liposome formulations in the delivery of anticancer drugs will be beneficial as they exhibited low drug leakage and efficient release.