This study aimed to investigate the influence of formulation factors on the physico-chemical properties of quercetin-loaded liposomes and optimize the preparation conditions. An experimental design with three factors and three levels was used. The variables were dipalmitoylphosphatidylcholine (DPPC) concentration, DPPC:Cholesterol (CHOL) molar ratio and quercetin (QU) concentration. The responses were the mean diameter, polydispersity index (PdI) and drug encapsulation efficiency. All samples were prepared according to the experimental design matrix consisting of 17 experiments. Results showed that DPPC concentration and DPPC:CHOL molar ratio had a strong influence on liposome size and PdI. Higher DPPC concentrations led to larger particles and PdI values, but a further increase of DPPC concentration produced a reduction in size and lower PdI. Increasing the lipid ratio caused a decrease in size, but a somewhat higher PdI. QU concentration only slightly influenced particle size and PdI. The degree of QU loading depended on the factors assessed. Increasing DPPC concentration and lipid ratio significantly enhanced QU entrapment. However, higher QU concentrations had a negative effect on drug entrapment. Based on this, an optimized formulation was determined, prepared and analysed. The overall results showed that DPPC concentration and lipid ratio were the main factors influencing particle size, while entrapment efficiency was affected predominantly by QU concentration.