Elastane blended apparel is one of the most preferred items by consumers with fashion interest due to its enhanced comfort and fit. The environmental impact and microfiber release due to elastane usage is often ignored due to its lower percentage in apparel. To address such a gap, this study aimed to quantify and characterize the microfiber release behavior of cotton/elastane knitted fabric. Cotton/Elastane blended knitted fabrics with three different proportions of Cotton/Elastane (98/2, 95/5, and 92/8) were considered for this analysis. Upon laundry and quantification, the results of the study showed that 98/2 Cotton/Elastane fabric released 21.04 ± 12.46 microfibers/sq.cm, whereas, 92/8 Cotton/Elastane fabric released 46.56 ± 6.21 microfibers/sq.cm. An increase in elastane proportion increased the overall emission of microfibers per unit area of fabric. The results also showed a higher contribution of elastane fibers in the total microfibers released. 13.40% of the total fibers released were elastane microfibers in the case of 98/2 Cotton/Elastane fabric, whereas, 92/8 Cotton/Elastane fabric released 19.60% of elastane microfibers. The elastane percentage of the fabric showed a significant positive correlation with total microfiber emission (r = 63%) and elastane microfiber emission (r = 62%). Repeated laundering results showed an overall reduction in microfiber emissions in subsequent washes. However, interestingly, an increase in the wash cycle increased the proportion of elastane microfibers in the total microfibers released. 92/8 Cotton/Elastane fabric released 20% of elastane microfibers in the first wash and the proportion increased to 36% in the 4th wash. In contrast to that, the release of cotton microfibers is noted to decrease with the number of laundry cycles. As far as the length of microfiber is considered, elastane microfibers are shorter than cotton microfibers. The length of elastane microfibers was higher in the initial wash (98/2 Cotton/Elastane fabric - 352.5 μm; 95/5 Cotton/Elastane fabric - 920 μm; 92/8 Cotton/Elastane fabric - 695 μm) and it is reduced with increment in the number of washes with a strong negative correlation of −0.88. A higher proportion of emissions and lower fiber length are the alarming negative impacts of elastane fibers in apparel. Based on this analysis, it is estimated that one square meter of fabric with a lower elastane percentage (2%) can release up to 2.81 × 104 microfibers into the environment at the first wash. The threatening issues of microfibers on aquatic life, particularly in terms of bioaccumulation and biomagnification, are alarming. Elastane blended fabrics should be given special attention because they can make the problem more serious by posing a risk of chemical leachates, such as bisphenols.