The invention discloses a laying hen oviduct ampulla epithelial cell culture and oxidative stress model establishment method. The cell culture method consists of: soaking a whole section of laying hen oviduct in PBS, and removing mesentery, connective tissue and blood; cutting off the oviduct ampulla, performing cleaning and then cutting it into tissue pieces; transferring the cleaned tissue pieces into collagenase IV for digestion; filtering the digestive juice, centrifuging the filtrate and discarding the supernate, adding a complete medium to resuspend the cell, and finally transferring the cell into a culture bottle to conduct cultivation. The oxidative stress model establishment method consists of: testing the heavy metal of different concentrations and the cell viability under different action time, and determining the action time point; designing different heavy metal concentration gradients according to the time point, determining cell apoptosis and other conditions, and establishing an oxidative stress model. The invention establishes the laying hen oviduct ampulla epithelial cell oxidative stress model for further exploration of the specific mechanism for egg white quality decline caused by the external environment.