Antisense oligonucleotides (ASOs) have promising therapeutic potential in oncotherapy. However, low stability and efficacy limit their application in the clinic. Cationic liposomes have been investigated as delivery vehicles for ASOs. Here, we report the synthesis and evaluation of an ASO delivery vehicle comprising cationic liposomes incorporating fatty acid-modified polyethylenimine. An oleic acid derivative of branched polyethylenimine (PEI-OA) and a linoleic acid derivative of branched polyethylenimine (PEI-LA) were synthesized and incorporated into liposomes. The PEI-modified liposomes were synthesized by an ethanol injection method with composition of PEI-modified lipid/Chol/TPGS. The properties of these liposomes, including cytotoxicity, cellular uptake, ASO target silencing activity, based on mRNA and protein downregulation, were investigated. LOR-2501, an ASOs targeting ribonucleotide reductase R1 subunit (R1) was used as the therapeutic cargo. The PEI-modified liposomes showed relatively compact particle size and excellent colloidal stability for at least 25 days. PEI-modified liposomes effectively delivered LOR-2501 into KB cells and efficiently induced down-regulation of R1 mRNA and protein. Compared with regular cationic liposomes, PEI-modified liposomes was more effective, reducing R1 mRNA and protein by ~10%.