The permeation of calcein across the phospholipid bilayer membrane is a key phenomenon in the detection system using liposomes as a sensor unit. The behavior of the calcein release from the liposome was analyzed by a first-order kinetic to obtain the permeability coefficient, Ps [cm/s]. The Ps value for the neutral liposome, prepared by zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was found to depend on both the diameter of liposome and the temperature. The membrane fluidity of the POPC liposome, evaluated by the hydrophobic probe, 1-(4-trimethyl-aminophenyl)-6-diphenyl-1,3,5-hexatriene, was also dependent on the liposome diameter and the temperature. The Ps values for various neutral liposomes under gel phase or liquid-crystalline phase were correlated with their membrane fluidity, although some data were a little scattered, possibly due to the lamellarity. It is therefore considered that the membrane fluidity dominates the permeability of calcein across the neutral phospholipid membrane. Based on the above results, the Ps value for liposomes immobilized on the solid surface is discussed.