As high temperature and relative humidity (RH) are the main environmental factors causing heat stress, the temperature-humidity index (THI) serves as an indicator of heat stress in livestock animals. This study aimed to determine the effects of heat stress on the laying performance, physiological responses, egg quality, and blood profile of laying hens by subjecting them to environmental conditions with varying THI levels (68-85) for 28 days. The indicators of laying performance, such as feed intake (-30%) and egg production rate (-11%), significantly decreased in the hens exposed to severe heat stress (33 °C, 66% RH) compared to those exposed to thermoneutral conditions (21 °C, 68% RH). Moreover, severe heat stress reduced the egg yolk color, eggshell thickness and strength, and Haugh units of the eggs produced by the laying hens. Furthermore, a significant increase in serum K+ and a decrease in Na+ levels were observed in the hens subjected to severe heat stress compared with those under thermoneutral conditions. Our results indicate that heat stress alters the physiological responses and metabolism of laying hens, resulting in a lower egg quality and production rate.