Blood oxygen saturation (SpOsub.2) is an essential indicator of a patient’s general condition. However, conventional measurement methods have some issues such as time delay and interference by ambient light. Improved measurement methods must be developed, and there are no reports on intraoral measurements of SpOsub.2 using wearable devices. Therefore, we aimed to establish an intraoral SpOsub.2 measurement method for the first time. Twelve healthy adults participated in this study. The following steps were taken: (1) to identify the optimal measurement location, mid-perfusion index (PI) values were measured at six places on the mucosa of the maxilla, (2) to validate the optimal measurement pressure, PI values were obtained at different pressures, and (3) using the proposed mouthpiece device, SpOsub.2 values in the oral cavity and on the finger were analyzed during breath-holding. The highest PI values were observed in the palatal gingiva of the maxillary canine teeth, with high PI values at pressures ranging from 0.3 to 0.8 N. In addition, changes in SpOsub.2 were detected approximately 7 s faster in the oral cavity than those on the finger, which is attributed to their proximity to the heart. This study demonstrates the advantage of the oral cavity for acquiring biological information using a novel device.