Abstract: Saline-alkaline water is a vast untapped resource for global aquaculture, holding great potential to alleviate the growing pressure of freshwater scarcity in aquatic food production. However, its high alkalinity-driven by elevated HCO₃⁻ and CO₃²⁻ ions-disrupts the ion and acid-base homeostasis of most fish species, leading to physiological stress, impaired growth, or even mortality, which severely limits its sustainable use in aquaculture. Leuciscus waleckii, a fish species endemic to northern China, has evolved extraordinary adaptability to extreme alkaline habitats (e.g., Dalai Lake with naturally high alkalinity), making it an ideal model organism for deciphering the physiological and molecular mechanisms of fish alkaline adaptation. This study aimed to investigate the effects of graded alkalinity stress on ion flux dynamics and acid-base balance regulation in embryos and gill tissues of two geographically isolated L. waleckii populations: the alkaline-adapted Dalai Lake (DL) population and the freshwater-acclimated Songhua River (SH) population. Experimental fish were exposed to three alkalinity gradients (0 mmol/L NaHCO₃ as control, CK; 30 mmol/L NaHCO₃, C_A30; and 50 mmol/L NaHCO₃, C_A50) for a 7-day acclimation period. Using Non-invasive Micro-test Technology (NMT)—a technique enabling real-time measurement of ion transport across biological membranes—we quantified the net fluxes of H⁺, Na⁺, and Cl⁻; assayed the activity of gill Na⁺/K⁺-ATPase (NKA, a key enzyme mediating ion pumping); and analyzed the mRNA expression of ion transport-related genes. H⁺ was continuously excreted from the embryonic epidermis and gills of both alkaline and freshwater-adapted L. waleckii under alkalinity stress. H⁺ flux was stable in gills but fluctuated greatly in embryos. Na⁺ and Cl⁻ were also secreted outward. The alkaline-adapted population exhibited stronger Na⁺ regulation than the freshwater population at both embryonic and juvenile stages, especially in gills(increase of 183%). Cl⁻ flux varied more obviously in the freshwater population. With elevated alkalinity, gill NKA activity and gene expression increased significantly, indicating strong ion and osmoregulatory ability. The expression of slc9a2, slc12a3, and slc12a2 was significantly upregulated with obvious population differences. The expression of slc4a4 and slc4a1 also changed markedly in the alkaline-adapted population. In conclusion, DL population has undergone adaptive evolution to high-alkaline environments, maintaining ion and acid-base balance through the coordinated regulation of NKA, NBC, and NCC transporters, and exhibits stronger alkaline tolerance than the SH population. This study provides critical insights into the mechanisms of fish adaptation to alkaline stress and lays a solid theoretical foundation for breeding alkali-tolerant fish strains to promote the sustainable development of saline-alkaline aquaculture.