Nanoscale ion-selective optodes (nano-ISOs) are versatile optical ion sensing tools with ionophores, ion exchangers, optical signal transducers, and sometimes plasticizers. Nano-ISOs were previously developed based on various nanoparticles with a topological solid core to contain the active sensing components. In this work, polymersomes, with a topologically hollow structure, were utilized as the matrix of nano-ISOs. Ca²⁺-selective polymersomes with high selectivity and a sub-micromolar detection limit were developed with a calcium ionophore and successfully used to determine the total calcium concentration in diluted human serum. With a sodium ionophore, Na⁺-selective polymersomes were also obtained, indicating that ionophore-based polymersomes are promising in forming a general ion-selective optical sensing platform.