Abstract: The test was to explore the optimum culture time for the everted gut sacs in broilers by the lactate dehydrogenase method. Using single factor test design, the duodenal, jejunum, ileum, intestine of 6 Three-yellow cocks were distributed randomly into conical flask with a broiler as a repeat and a intestinal segments as 6 repeats. Then at 0, 20, 40, 60 and 80 min, 100 uL culture solution in each conical flask was taken with a pipette gun to analyze the activity of lactate dehydrogenase. The result showed that, the activity of lactate dehydrogenase in culture solution was increased slowly from 0 min to 60 min and the rising range was faster after 60 min. The optimum culture time for the everted gut sacs in broilers was 40-50 min.