RNA interference has significant potential as an important tool in biology and medicine. For the development of highly efficient non-viral siRNA carriers, hydrophobic modification on cationic polymers has received increasing attention. Here, we described the screening of efficient siRNA carriers in a library of hydrophobically modified polyethyleneimines (PEIs). Cationic PEI was grafted with hydrophobic ligands including alkanes, cycloalkanes and fluoroalkanes. These modifications significantly improved the gene knockdown efficacy of PEI. Among the functionalized polymers, the fluoroalkylated PEIs showed higher efficacies and lower toxicities than the alkylated and cycloalkylated ones. The top performing polymers in the library efficiently knocked down several target genes in different cell lines without off-target effects. The revealed structure-activity relationships of alkylated, cycloalkylated and fluoroalkylated PEIs in this study provide a new insight into the design of highly efficient and non-toxic siRNA carriers.