The number and spacing of B-cell epitopes on antigens have a profound impact on the activation of B cells and elicitation of antibody responses, the quantitative aspects of which may be utilized for rational design of vaccines. Ni-chelating liposomes have been widely used as protein carriers in experimental studies of vaccine delivery, owing to the convenience and versatility of this conjugation chemistry. However, the epitope number per particle as well as the stability of protein conjugation on liposomes remain far less characterized. Here we have developed quantitative methods to measure the average spatial density of proteins on liposomes using both ensemble and single-molecule techniques and demonstrated their utility using liposomes conjugated with native proteins of two different sizes. These studies revealed that the initial density of protein conjugation on Ni-chelating liposomes can be finely controlled, but the density can decrease over time upon dilution due to the noncovalent nature of Ni-chelation chemistry. These results indicate that an alternative method other than the Ni-chelation chemistry is needed for stable conjugation of epitopes onto liposomes and also suggest a general strategy that can be used to precisely regulate the epitope density on liposomes for B-cell antigen delivery.