In order to describe temperature-dependent cell osmotic behaviors in a more reliable method, a novel mathematical mass transfer model coupled with dynamic temperature change has been established based on the combination of a time domain to temperature domain transformation equation and a constant temperature mass transfer model. This novel model is numerically simulated under multiple temperature changing rates and extracellular osmolarities. A microfluidic system that can achieve single-cell osmotic behavior observation and provide dynamic and swift on-chip temperature control was built and tested in this paper. Utilizing the temperature control system, the on-chip heating processes are recorded and then described as polynomial time-temperature relationships. These dynamic temperature changing profiles were performed by obtaining cell membrane properties by parameter fitting only one set of testing experimental data to the mathematical model with a constant temperature changing rate. The numerical modeling results show that predicting the osmotic cell volume change using selected dynamic temperature profiles is more suitable for studies concerning cell membrane permeability determination and cryopreservation process than tests using constant temperature changing rates.