IntroductionEstablishing the reference interval for pulse oxygen saturation (SpO2) is essential for sensitively identifying neonatal hypoxaemia due to various causes. However, the reference interval for high altitudes has not yet been established, and existing studies have many limitations. This study will aim to establish the reference interval for various high altitudes and determine whether preductal and postductal measurements at the same altitude vary.Methods and analysisThis is a multicentre, open, cross-sectional study, which will begin in February 2022. Approximately 2000 healthy full-term singleton neonates will be recruited from six hospitals (altitude ≥2000 m) in Qinghai Province, China. The participating hospitals will use a uniform pulse oximeter type. The measurements will be performed between 24 hours after birth and discharge. During the measurement, the neonate will be awake and quiet. Preductal and postductal measurements will be performed. The measurement time, site and results will be recorded and input, along with the collected basic information, into the perinatal cloud database. We will carry out strict quality control for basic information collection, measurement and data filing. We will perform descriptive statistics on the distribution range of the collected data, determine the lower limit value of the reference interval for each hospital and the corresponding altitude, perform curve fitting for the lower limit value, use the altitude as a covariate for the function corresponding to the fitted curve, establish the prediction equation and ultimately determine the reference intervals of each high altitude location.Ethics and disseminationOur protocol has been approved by the Medical Ethics Committee of all participating hospitals. We will publish our study results in academic conferences and peer-reviewed public journals.Trial registration numberNCT05115721.