Liposomes are widely used as drug delivery systems. However, inefficient delivery limits their application in serum containing systems. In this study, two cholesterol derivatives were synthesized and incorporated into liposomes. The charge influence on drug delivery was investigated. The results indicated that the positively charged liposomes showed a higher delivery efficiency for drugs with both small molecular weight (DOX, doxorubicin) and macromolecular weight (polyethylene glycol 6000 conjugated with rhodamine B, PEG-RhB) into cells, compared with neutral liposomes even in the presence of serum. The cytotoxicity of the positive liposomes was lower than that of DOTAP (N-(1-(2,3-dioleoyloxy) propyl-N,N,N-trimethylammonium mesylate) liposomes. Moreover, results from confocal microscopy indicated that the positive DOX-liposomes underwent a quick binding process onto the cell membrane, followed by drug uptake by the cell. In vivo experiments also revealed that the positive DOX-liposomes had a higher drug delivery ability into rat retina than the neutral ones.