A person spends about a third of their life sleeping, and high sleep quality is very important for health. Environmental factors are one of the most important factors affecting sleep quality, and indoor carbon dioxide (CO₂) concentration while sleeping has a significant effect on sleep quality. In an indoor bedroom with no open windows and no fresh air system, different numbers of people sleeping will lead to changes in indoor CO₂ concentration. In order to study the changes in sleep quality caused by differences in CO₂ concentration, experimental research was performed. Objective sleep quality data are collected with polysomnography (PSG) and a subjective questionnaire. The sleep quality of the subjects is tested under three different CO₂ concentration levels; the average carbon dioxide concentration of three conditions is 680, 920, and 1350 ppm, which simulate a room with 1, 2, and 3 people sleeping, respectively. Other environment parameters are controlled as follows: test environment temperature is 26 ± 0.5 °C, relative humidity is 50 ± 5%, there is no obvious heat source in the test room, and the radiation temperature and air temperature difference is less than 1 °C. A total of 30 subjective tests were carried out with 10 subjects; the test lasted more than one month. The data subsequently underwent statistical analysis to determine the influence of CO₂ concentration on sleep quality. The results show that as the CO₂ concentration level increased, the sleep quietness and satisfaction of the subjects gradually decreased, the sleep duration gradually decreased, and symptoms such as throat discomfort, dyspnea, dry and itchy skin, difficulty falling asleep, difficulty waking up, congested nose and bad air smell become more obvious. The PSG test results showed that CO₂ concentration has a significant impact on the proportion of the N3 period. According to the group of CO₂ concentration conditions, the mean of the N3 period proportion under the conditions of one person, two persons, and three persons is 20.4%, 17.3%, and 14.4%, respectively. Finally, there was also an increase in turning over or awakening during sleep, indicating that sleep quality was reduced under higher CO₂ concentrations.