In this work, thermoresponsive diblock copolymers, poly[2-(2-methoxyethoxy) ethyl methacrylate]-b-poly(2-hydroxyethyl methacrylate) (PMEO(2)MA-b-PHEMA) with low polydispersity were synthesized by atomic transfer radical polymerization(ATRP). Low molecular weight (LWM) polyethylenimine (PEI, 1200Da) was then grafted to 1,1'-carbonyldiimidazole (CDI)-activated PMEO(2)MA-b-PHEMA to fabricate PEI-g-(PMEO(2)MA-b-PHEMA) (PEIMH) copolymer vectors. The LCSTs of PEIMHs with 3 and 8 grafted PEI side chains, separately termed as PEIMH-1 and PEIMH-2, were 32.5 and 38.7 degrees C in PBS solution. Variable temperature agarose retardation, Zeta potential and time-resolved fluorescence assays were performed to elucidate the temperature sensitive DNA condensation. It showed that DNA was condensed more efficiently by PEIMH, and the collapse of PMEO(2)MA chains led to more exposure of surface positive charges of PEIMH-1/pDNA complexes while temperature was above LCST. Variable temperature time-resolved fluorescence measurement of lifetimes of bound and free ethidium bromide (EB) unveiled that the population of EB at different states was dependent on temperature. At a temperature above LCST, the collapsed PMEO(2)MA polymer chains squeezed the loosely bound EB out of complex to become free species; thereby DNA was more tightly packaged by PEIMH-1. Temporary cooling was shown to improve the transfection efficiency of PEIMH-1 in COS-7 and HEK293 cell lines. The variable temperature protocol is more efficient in improving gene expression level in HEK293 cells. The transfection efficiency was equivalent or superior to that of PEI25K at an optimal weight ratio of vector/DNA. Furthermore, the cytotoxicity of PEIMH-1 was considerably lower than that of control PEI25K.