Abstract: This study investigated the growth performance of monosex-cultured all-female large yellow croaker (Larimichthys crocea) and their response to continuous cooling stress. The experimental group consisted of all-female fish (body weight: 14.09±2.72 g), while the control group included naturally reproduced offspring, comprising females (14.01±2.52 g) and males (13.87±3.17 g). Over a period of 120 days, growth performance was compared across groups, and the effects of cooling stress on tissue structure, immune responses, and intestinal microbiota were analyzed. At days 30 and 60, no significant differences in body weight were observed among groups. By day 120, the all-female group exhibited significantly higher body weight and weight gain rate than those of control group males, with no differences compared to the females. Under continuous cooling stress, all groups exhibited vacuolation in gill cells, severe gill filament deformation, intestinal cell fusion, goblet cell swelling, mucosal necrosis, and shedding. Enzyme activities (SOD, CAT, GSH-Px) and MDA content initially increased significantly, followed by a significant decrease. At 6°C, SOD and CAT activities in the gills of the all-female group were significantly lower than those of the control group. Similarly, in the intestine, CAT and GSH-Px activities, along with MDA content, were lower in the all-female group. IL-6 gene expression in the all-female group was also significantly lower than that in the control group. Intestinal microbiota analysis showed that low-temperature stress altered the relative abundance of dominant phyla and genera in the all-female group and control group females, with no significant effect on males. Microbiota network structure changes were observed across all groups. In conclusion, monosex all-female large yellow croaker maintained rapid growth under the monosex farming model. However, low-temperature stress induced tissue damage, oxidative stress, inflammatory responses, and microbiota network alterations in all groups. This study serves as a valuable reference for advancing monosex farming research for large yellow croaker.