Chemical defoliants are widely used in cotton (Gossypium L.) to accelerate leaf abscission and boll maturation, as well as, to facilitate mechanical harvesting. The current study was conducted to determine the interactive effect of cotton cultivars and spraying time of defoliant on defoliation, boll opening, fibre yield and quality. An experiment was performed with four cultivars and three defoliant spraying time during 2019 and 2020 in split plot design with three replications. At harvest, the defoliation and boll opening rate of all treatments after spraying defoliant was 94.6 and 85.4%, while the blank control (water) was 73.9 and 79.1%, respectively. After spraying defoliant, the effects of defoliation rate, boll opening rate, fibre yield and quality were different among cultivars, indicating that different cultivars had different responses to defoliant. Among them, L7619 was the most sensitive to defoliant, with the average defoliation rate of 95.6% and a seed cotton yield reduction of 882.9 kg/ha. Among the different time of applications, late spraying (17 September, B3) of defoliant recorded the highest defoliation rate (97.3%), boll opening rate (89.8%), seed cotton yield (3991 kg/ha) and steadily increased the fibre strength by 0.59 cN/tex compared with the control. Late spraying of defoliant had little or even no adverse effect on the remaining fibre quality traits (length, uniformity, micronaire and elongation). In general, these results suggested that the appropriate time for spraying defoliant can be determined based on the sensitivity of the cotton cultivar, the weather conditions at the field and the harvest time.