The previous research on folate-conjugated liposomes has shown inconsistent results due to various factors such as the diverse characteristics of the ligand the presence of polyethylene glycol (PEG) coating, linker length, and ligand density. HSPC, cholesterol, and DSPE-PEG2k using the thin-film hydration method. Instead of DSPE-PEG2k alone, a mixture of DSPE-PEGn-Folate (n = 2k, 5k, and 10k) and DSPE-PEG2k. Doxorubicin-loaded non-targeting liposomes (DOX/NTLs) and folate-conjugated liposomes (DOX/FL) were then evaluated in folate receptor–overexpressing KB cells to investigate nanoparticle distribution and the pharmacological efficacy of the encapsulated drug both in vitro and in vivo. The in vitro studies showed no significant differences between formulations with varying PEG linker lengths. However, in vivo studies revealed that longer PEG chains enhanced tumor targeting. In particular, the DOX/FL-10K group reduced tumor size by more than 40% compared with DOX/FL-2K and DOX/FL-5K groups, demonstrating improved anticancer efficacy and tumor-targeting ability. These findings suggest that increasing the length of the PEG linker can enhance tumor targeting in vivo and improve the antitumor efficacy of the encapsulated drug.