We developed an integrated microfluidic platform for instantaneous flow and localized temperature control. The platform consisted of a flow-focusing region for sample delivery and a cross-junction region embedded with a microheater for cell trapping and localized temperature control by using an active feedback control system. We further used it to measure the membrane transport properties of Jurkat cells, including the osmotically inactive cell volume (Vb) and cell membrane permeabilities to water (Lp) and to cryoprotective agent (CPA) solutions (dimethyl sulfoxide (DMSO) in this study) (PS) at various temperatures (room temperature, 30 °C, and 37 °C). Such characteristics of cells are of great importance in many applications, especially in optimal cryopreservation. With the results, the corresponding activation energy for water and CPA transport was calculated. The comparison of the results from the current study with reference data indicates that the developed platform is a reliable tool for temperature-dependent cell behavior study, which provides valuable tools for general cell manipulation applications with precise temperature control.