||Electroendocytosis (EED), i.e. electric-field-induced endocytosis-like process, is a technique to facilitate molecules delivery to cells using a pulsed electric field train (PEF). PEF enhances the cell plasma membrane invagination and fission via endocytotic vesicles and herein benefits exterior molecules uptake. In this study, the EED phenomenon of HeLa cells was conducted from large-scale experimental study using a novel micro system and in-situ fluorescence microscopy. The novel micro system consists of integrated microelectrodes, micro temperature sensors and a thermoelectric device was fabricated and applied, for the first time, to investigate the effects of multiple electric parameters, temperature and chemicals on EED efficiency, dynamics and cellular viability at the single-cell level. The EED uptakes of lipid labeling probes (FM4-64) and macromolecules were characterized by the average intracellular fluorescent intensity and endocytosis vesicle number from a large number of individual cells with the help of digital fluorescence microscopy. Effects from PEF (electric field strength, pulse duration, total electric treatment time) were examined. The EED efficiency as a function of electric field strength was shown to be biphasic with a transition EED electric field (Et,EED). From scaling analysis, the electric field’s exponents of EED and electroporation (EP) are 2.19±0.32 and 6.21±1.67, respectively. PEF has been demonstrated to enhance endocytosis in terms of uptake rate and activity persistence. In addition, the existence of a general critical temperature (Tc) of ca 16-18 ºC for HeLa cells EED was determined. PEF was suggested to enhance endocytosis not by affecting membrane lipid phase transition, but possibly owing to stimulate the thermal fluctuation of cell membrane. Besides, EED experiments using chemical inhibitors (Demecolcine and Cytochalasin D) illustrated its dependence on cytoskeleton and its unique mechanisms distinct from EP. Finally, an energy model was proposed to describe the mechanisms of EED, which are due to PEF induced membrane fluctuation and invagination.