Fever screening is an effective method to detect infectors associated with different variants of coronavirus disease 2019 (COVID-19) based on the fact that most infectors with COVID-19 have fever symptoms. Non-contact infrared thermometers (NCITs) are widely used in fever screening. Nevertheless, authoritative data is lacking in defining “fever” at different body surface sites when using NCITs. The purpose of this study was to determine the optimal diagnostic threshold for fever screening using NICTs at different body surface sites, to improve the accuracy of fever screening and provide theoretical reference for healthcare policy. Participants (n = 1860) who were outpatients or emergency patients at Chengdu Women's and Children's Central Hospital were recruited for this prospective investigation from March 1 to June 30, 2021. NCITs and mercury axillary thermometers were used to measure neck, temple, forehead and wrist temperatures of all participants. Receiver operating characteristic curves were used to reflect the accuracy of NCITs. Linear correlation analysis was used to show the effect of age on body temperature. Multilinear regression analysis was used to explore the association between non-febrile participant's covariates and neck temperature. The mean age of participants was 3.45 ± 2.85 years for children and 28.56 ± 7.25 years for adults. In addition 1,304 (70.1%) participants were children (≤12), and 683 (36.7%) were male. The neck temperature exhibited the highest accuracy among the four sites. Further the optimal fever diagnostic thresholds of NCITs at the four body surface measurement sites were neck (36.75 °C, sensitivity: 0.993, specificity: 0.858); temple (36.55 °C, sensitivity: 0.974, specificity: 0.874); forehead (36.45 °C, sensitivity: 0.961, specificity: 0.813); and wrist (36.15 °C, sensitivity: 0.951, specificity: 0.434). Based on the findings of our study, we recommend 36.15, 36.45, 36.55, and 36.75 °C as the diagnostic thresholds of fever at the wrist, forehead, temple and neck, respectively. Among the four surface sites, neck temperature exhibited the highest accuracy.