In vitro gene delivery, polyethyleneimine (PEI) has been described as one of the most efficient nonviral vector. Herein the formation mechanism of PEI/DNA complexes is elucidated. The transition phase of "bead-on-string" structure in the formation of complexes was supposed to exist through spectroscopy, electrophoresis and transmission electron microscopy (TEM) technology. The construction of PEI/DNA complexes is related closely to the characteristics of PEI and DNA plasmid. As well as the dominant electrostatic effects, the nonelectrostatic interactions were thought to be partially responsible for the presence of PEI/DNA complexes even in the high ionic strength. The surface charge of complexes particles increased with the N/P ratio, but the absolute value of zeta potential was lower at the N/P ratio of 8 and 12, perhaps attributed to the use of larger DNA plasmid. As a result, the repulsion between particles was decreased and prone to aggregate to the structure like a clustered grape-string in the solution. Interestingly, contrast to the formation behavior of complexes, the PEI/DNA complexes aggregated primarily due to hydrophobic interactions while electrostatic attractions play a little role in the complexes particles aggregation in different concentrations of salt solutions. Comparable transfection efficiency in HepG2 cells was observed for the Lipofectamine 2000 and PEI/DNA complexes at the N/P ratio of 12, and showed that larger or aggregable complexes could transfect the cells in some different mechanisms.