To measure the 24-h intraocular pressure (IOP) by Icare PRO rebound in healthy and primary open-angle glaucoma (POAG) eyes and compare it with non-contact tonometry (NCT). Thirty POAG patients, who were under IOP-lowering treatment, and 30 healthy subjects were included. Participants were hospitalized overnight for the 24-h IOP measurement. IOPs were measured by Icare PRO and NCT according to a standard protocol every 2h during 24h. The 24-h IOP curve and IOP-related parameters were compared between Icare PRO and NCT groups in POAG and healthy eyes. The IOPs measured by Icare PRO in habitual position increased notably at 22:00 in the normal group and at 20:00 in the POAG group, reached peak at 0:00, stayed high until 4:00, and then decreased in both groups (all p < 0.05). The POAG patients had higher mean 24-h IOP, peak IOP, IOP fluctuation, and greater IOP change from supine to sitting position in the nocturnal period than those in the normal subjects even after adjusting for eyes, age, gender, CCT, and axial length (all p < 0.05). The Icare PRO provides a well-tolerated approach for 24-h IOP monitoring in habitual position. Twenty-four-hour IOP in habitual position is more sensitive for detecting high nocturnal IOP peaks and greater IOP fluctuation for POAG patients.