Disulfide cross-linked polyethylenimines (PEI(X)-SS(Y), where X refers to the molecular weight of raw PEI, and Y refers to the thiolation degree) were prepared in two steps: First, thiol groups were introduced on a raw polyethylenimine (PEI) by the amine-induced ring-opening reaction of thiirane. Second, thiol groups were oxidized by DMSO to form the disulfide cross-links. The cross-linked PEI(800)-SS(Y) polymers with a moderate thiolation degree (PEI(800)-SS(2.6,) PEI(800)-SS(3.5), and PEI(800)-SS(4.5)) could form compact polyplexes with a size of 200-300 nm at an adequate N/P ratio. In contrast, those with a too low or too high thiolation degree (Y below 2.6 or above 4.5) formed much looser polyplexes with a size above 600 nm. The polyplexes of PEI(X)-SS(3.0-4.0) series (X = 800, 1800, and 25,000) formed small particles with a size below 400 nm at a wide range of N/P ratios. Efficiency of the cross-linked PEIs as gene vectors was evaluated in vitro by transfection of pGL3 to HeLa, COS7, 293T, and CHO cells. The efficiency is disulfide content and molecular weight dependent. The PEI(800)-SS(Y) series with an adequate thiolation degree between 2.6 and 4.5 have relatively lower cytotoxicity and higher gene transfection efficiency than 25 KDa PEI. The polymers with very low or very high thiolation degrees were unable to form compact polyplexes and had very poor transfection efficiency. A suitable molecular weight of raw PEI is also essential to obtain a highly efficient disulfide cross-linked PEI gene vector. Among the three raw PEIs of different molecular weights tested (800 Da, 1800 Da, and 25 KDa), the cross-linked polymer prepared from 800 Da PEI that has the lowest molecular weight gave the best results.