Protein-rich aqueous samples such as milk and plasma usually require complex sample preparation steps prior to instrumental analysis. This study proposed a novel cotton fiber-supported liquid extraction (CF-SLE) method for convenient sample preparation. Natural cotton fiber was directly loaded into a syringe tube to conveniently construct the extraction device. No filter frits were required due to the fibrous feature of the cotton fibers. The cost of the extraction device was less than 0.5 CNY, and the costly syringe tube could be easily reused to decrease the cost further. Extraction used a simple two-step protocol: protein-rich aqueous sample loading and elution. Emulsification and centrifugation steps involved in the classic liquid–liquid extraction were avoided. As a proof-of-concept study, the glucocorticoids in milk and plasma were extracted with satisfactory extraction recoveries. Coupled with liquid chromatography-tandem mass spectrometry, a sensitive quantification method was established with excellent linearity (R2 > 0.991) as well as good accuracy (85.7–117.3%) and precision (<14.3%). This system is simple, low-cost, reproducible, and easy to automate. Thus, the proposed CF-SLE method is promising for the routine sample preparation of protein-rich aqueous samples prior to instrumental analysis.