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In order to measure the real-time variations in body temperature with increasing ambient temperature (AT) at different relative humidity (RH) levels, 60 Jinghong laying hens (35-wk-old) were raised in 3 controlled climate chambers (10 cages with 2 birds per chamber). The RH was fixed at one of 3 levels comprising 35, 50, or 85%, and the AT was increased gradually by 1 degree per 0.5 h from 18 to 35°C in the 3 chambers. The core temperature (CT) and surface temperature (ST) of the hens, as well as the AT in the 3 chambers were recorded at 3 min intervals using mini temperature data loggers. The data were analyzed with a broken-line model to determine the inflection point temperature (IPT, the certain AT above which the body temperature of the hens started to change). The experiment was repeated 3 times on 3 d. The IPTs of the laying hens were 23.89 and 25.46°C based on ST and CT at 50% RH, respectively, which indicated that the upper critical temperature of the thermoneutral zone of hens may be a specific temperature between 23.89°C and 25.46°C. The IPTs of the laying hens were 24.11 and 25.20°C based on ST and CT at RH 35%, respectively, and 21.93 and 24.45°C at RH 85%. The RH significantly affected the IPT of ST (P < 0.001). The IPTs were higher at 35 and 50% RH than that at 85% RH (P < 0.05). The coefficients of variation for the IPTs between individual hens were 2.96 to 4.51, and coefficients of variation for the IPTs for the same bird measured on 3 d were 0.69 to 1.59, thereby indicating that this method for estimating the IPTs of hens is stable and repeatable, although more samples are needed. In conclusion, our results indicate that analyzing the real-time variation in body temperature with increasing AT is a reliable method for estimating the IPT to provide an important reference for regulating the temperature in poultry houses.