Abstract: To comprehensively assess the alkali tolerance of grass carp “Husu No.1”, this study examined and analyzed the significant physiological response parameters and related gene expressions in gills of grass carp under varying NaHCO₃ alkalinity gradients: low (10 mmol/L, AW-10), medium (30 mmol/L, AW-30), and high (50 mmol/L, AW-50) over a 7-day period. The findings revealed that in terms of ammonia nitrogen metabolism, as alkalinity increased, the content of blood ammonia exhibited an ascending trend, with the AW-50 group having the highest content. However, there was no significant difference (P>0.05) between the low and medium alkalinity groups and the freshwater control group. The content of serum urea nitrogen was lower than that of the freshwater control group. The activity of glutamate dehydrogenase (GDH) and the expressions of rhbg, rhcga, and glud1a in the gills of the medium and high alkalinity groups were upregulated, suggesting that grass carp might carry out ammonia detoxification by enhancing the high expression of ammonia transport genes in the gills and activating the GDH pathway, thereby stabilizing the stability of blood ammonia at medium and low alkalinity. Regarding the immune response, although the numbers of three types of immune cells (white blood cells, lymphocytes, and neutrophils) in different alkalinity groups did not significantly differ from those in the freshwater control group, the numbers of these three immune cells showed an increasing trend with the increase in alkalinity, with the AW-50 group showing the greatest increase. The expression of immune-related gene c3 was significantly elevated across all alkalinity groups, while il-1β was significantly increased in the high alkalinity group, tnf-α was significantly increased in the middle alkalinity group, and more serious inflammatory reactions occurred in the middle and high alkalinity groups. In the aspect of ion and osmotic pressure regulation, there was no significant difference (P>0.05) in the contents of four serum ions (Na⁺, Cl^-, K⁺ and Ca²⁺) across the alkalinity groups compared to the freshwater control. However, the content of Na⁺ showed a decreasing trend with the increase in alkalinity, with the lowest content in the AW-50 group. Although the activity of sodium-potassium ATPase (NKA) in the gills in the AW-30 and AW-50 groups was significantly lower than that in the freshwater control group (P<0.05), the corresponding genes ATP1a1b and ATP1a3b were extremely significantly upregulated in the AW-30 group, suggesting that gene expression might be related to the lag in protein synthesis. In terms of blood oxygen transportation, the expressions of gill hemoglobin formation genes hbaa2 and hbba1 of grass carp under normoxia in different alkalinity groups were significantly inhibited. However, the number of red blood cells, hemoglobin content, and serum iron content increased with the increase in alkalinity, reaching the highest in the AW-50 group, indicating symptoms of hypoxia. Through comprehensive analysis, when the alkalinity is ≤30 mmol/L, grass carp can basically maintain physiological homeostasis. However, when alkalinity exceeds this critical value, the risk of death due to ammonia poisoning and hypoxia significantly increases. This study concludes that the alkali tolerance of grass carp is moderate, with the potential for breeding new alkali-tolerant varieties, and it is suitable for breeding and culture in water bodies with alkalinity ≤30 mmol/L.