In order to explore the regulation mechanism of alkali resistance of Leuciscus waleckii, a dominant natural species in an alkaline water environment, gill cells of Leuciscus waleckii were cultured by trypsin digestion in vitro, and the suitable conditions for primary and subculture cell culture were determined. The tolerance experiment was carried out to demonstrate its growth characteristics in an alkaline environment. The experimental results show that the primary gill cells of Leuciscus waleckii can be cultured in a DMEM medium containing 10% Fetal bovine serum(FBS) at 15 ℃, which can obtain a stable and good culture effect. It can be passaged within 36-72 h, and the stably passaged cells are named LWG cells. During subculture, LWG cells grew and metabolized vigorously at 36 h, which can be used for subsequent experiments. In alkaline water habitat, gill cells will start the osmotic pressure stress response mechanism to alleviate the damage caused by alkali ions to fish. Therefore, an alkali stress experiment on LWG cells was carried out and their cell activity and function were tasted. It was found that when LWG cells tolerated for three hours the concentration of 25-50 mmol/L NaHCO₃, proliferation rate was about 60% and apoptosis occurred. It can be inferred that LWG cells have strong osmotic alkalinity tolerance and promote apoptosis under carbonate alkalinity stress of 25-50 mmol/L. This will provide stable experimental materials for exploring osmotic stress response mechanisms and verifying related gene functions under alkali stress.