Active targeting has been hailed as one of the most promising strategies to further enhance the therapeutic efficacy of liposomal nanomedicines. Owing to the critical role of ligand density in mediating cellular uptake and the intrinsic heterogeneity of liposomal formulations, precise quantification of the surface ligand density on a single-particle basis is of fundamental importance. In this work, we report a method to simultaneously measure the particle size and the number of ligands on the same liposomal nanoparticles by nanoflow cytometry. Then the ligand density for each individual liposome can be determined. With an analysis rate up to 10 000 particles per minute, a statistically representative distribution of ligand density could be determined in minutes. By utilizing fluorescently labeled recombinant receptors as the detection probe against the conjugated ligands, only those available for cell targeting can be exclusively detected. The influence of ligand input, conjugation strategy, and the polyethylene glycol spacer length on the available ligand density of folate-modified liposomes was investigated. The correlation between the available ligand density and cell targeting capability was assessed in a quantitative perspective for liposomes modified with three different targeting moieties. The optimal ligand density was determined to be 0.5-2.0, 0.7, and 0.2 ligand per 100 nm for folate-, transferrin-, and HER2-antibody-conjugated liposomes, respectively. These optimal values agreed well with the spike density of the natural counterparts, viruses. The as-developed approach is generally applicable to a wide range of active-targeting nanocarriers.