More than 15 million tons of textile waste are generated every year in North America, but less than one fifth are recycled. The disposal of textile waste via landfill or incineration causes environmental problem and also represents a waste of useful resources. In this work, we explored the possibility to extract cellulose nanocrystals (CNCs) from textile waste through two methods, namely sulfuric acid hydrolysis and three-step oxidization. CNCs with cellulose Ⅰ crystalline structure and rod-like shape were successfully obtained. The aspect ratios of CNCs prepared from acid hydrolysis and oxidization were 10.18 and 16.17, respectively. Their application as reinforcing agent of soybean protein isolate (SPI) films was evaluated. With the addition of 20% CNCs, the composite films maintained the high transparency, while their water barrier property, tensile strength, and Young’s modulus were significantly improved. This research demonstrates a promising approach to recycle textile waste, and more value-added applications based on the derived CNCs could be expected.