Osmotic pressure can break the fluid balance between intracellular and extracellular solutions. In hypo-osmotic solution, water molecules, which transfer into the cell and burst, are driven by the concentrations difference of solute across the semi-permeable membrane. The complicated dynamic processes of the intermittent burst have been previously observed. However, the underlying physical mechanism has yet to be thoroughly explored and analyzed. Here, the intermittent release of inclusion in giant unilamellar vesicles was investigated quantitatively, applying the combination of experimental and theoretical methods in the hypo-osmotic medium. Experimentally, we adopted highly sensitive EMCCD to acquire intermittent dynamic images. Notably, the component of the vesicle phospholipids affected the stretch velocity, and the prepared solution of the vesicle adjusted the release time. Theoretically, we chose equations numerical simulations to quantify the dynamic process in phases and explored the influence of physical parameters such as bilayer permeability and solution viscosity on the process. It was concluded that the time taken to achieve the balance of giant unilamellar vesicles was highly dependent on the structure of the lipid molecular. The pore lifetime was strongly related with the internal solution environment of giant unilamellar vesicles. The vesicle prepared in viscous solution accessed visualized long-lived pore. Furthermore, the line tension was measured quantitatively by the release velocity of inclusion, which was in the same order of magnitude as the theoretical simulation. In all, the experimental values well matched the theoretical values. Our investigation clarified the physical regulatory mechanism of intermittent pore formation and inclusion release, which had an important reference for the development of novel technologies such as gene therapy based on transmembrane transport as well as controlled drug delivery based on liposomes.