This study was conducted to investigate the effects of dietary NaHCO₃ on performance, hematological indices, serum biochemical parameters and blood gas indices of laying hens, and to identify the safe dose range of sodium bicarbonate (NaHCO₃) in the diets of laying hens. Nine hundred 21-week-old Hy-Line brown laying hens were randomly assigned to 5 groups with 6 replicates per group and 30 birds per replicate. Laying hens were fed a basal diet with 0 (control group), 1, 5, 25 and 50 g/kg NaHCO₃ for 10 weeks, respectively. The results showed as follows: the performance of laying hens was not influenced by dietary NaHCO₃ supplemental level at ≤5 g/kg (P>0.05), but the laying rate, egg mass, feed intake, and average egg weight were significantly reduced (P<0.05 or P<0.01) and the ratio of broken and soft eggs was significantly increased (P<0.05) by dietary NaHCO₃ supplemental level at ≥25 g/kg NaHCO₃. In addition, the concentrations of HCO₃^- and total CO₂, partial pressure of CO₂, base excess, and pH in blood of laying hens were increased with increasing NaHCO₃ supplemental level, and significantly higher than those in the control group (P<0.05 or P<0.01) except that 1 g/kg NaHCO₃ did not affect pH and partial pressure of CO₂ (P>0.05). Moreover, the supplementation of NaHCO₃ did not affect the number of red blood cells and white blood cells, the activities of alkaline phosphatase, glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase and the concentrations of total protein, albumin, total bilirubin in serum of laying hens (P>0.05). However, blood hematocrit and the concentrations of hemoglobin in blood, creatinine and usea nitrogen in serum (P<0.05 or P<0.01) were markedly increased and the concentration of blood glucose in serum (P<0.05) was also significantly decreased by the supplementation of 25 to 50 g/kg NaHCO₃. Furthermore, the number of platelet in blood of laying hens in 50 g/kg NaHCO₃ group was notably lower than that in the other groups (P<0.05). It is concluded that excessive supplementation of NaHCO₃ (≥25 g/kg) can cause the decrease of performance, change of blood gas indices, and disorder of physiological functions of laying hens. However, the supplementation of NaHCO₃ at <5 g/kg can support the performance and assistant to maintain hematological levels in the physiological ranges of laying hens. Therefore, it is suggested that the safe dose of NaHCO₃ in diets of laying hens should be lower than 5 g/kg.