[Objective] To explore the mechanism of the effects of abscisic acid (ABA) on the photosynthetic effect and ultrastructure of cotton leaves at flowering and bolling stage, and to provide theoretical basis for the regulation of ineffective water loss of cotton in southern Xinjiang by ABA. [Methods] The plant of Tahe No. 2 was treated with ABA at low concentration (50 μmol/L), high concentration (100 μmol/L) and water (control). Photosynthetic pigment content, photosynthetic gas exchange parameters, stomatal characteristics and ultrastructure of the leaves were determined. [Results] Compared with the control group, after 1 day of spraying ABA, the stomatal density, shape index and chloroplast area of cotton leaves were increased, while the chlorophyll content, net photosynthetic rate and transpiration rate were decreased, and the effect of high concentration of ABA treatment was more obvious. After spraying ABA for 4 days, the stomatal density of cotton leaves in high concentration treatment decreased and the shape index increased, while that in low concentration treatment increased. [Conclusion] After spraying ABA, the stomatal closure of cotton leaves was affected by adjusting stomatal density and morphological index, resulting in the decrease of chlorophyll content, but the chloroplast structure was automatically adjusted to offset this adverse effect, and high concentration treatment had a better effect on reducing the ineffective water loss of cotton.