In this study, the authors used an enzyme called papain sourced from the Carica Papaya to improve the comfort and water-absorbing properties of a fabric made from a blend of polyester and cotton (65/35). The experiment was designed using the Box Behnken method to determine the most important variable and the best levels of parameters. The focus was on testing the wettability, moisture regain, and surface characteristics of the material. The results showed that all the comfort properties of the fabric improved after treatment with papain enzyme. After testing different parameters, the best conditions for treating the fabric with papain enzyme were determined to be a temperature of 30 °C, a papain concentration of 14%, and a treatment time of 50 min. Under these optimized conditions, the moisture regain and wettability of the polyester/cotton blend fabric treated with papain enzyme improved to 1.9 ± 0.02% and 6 cm capillary rise (measured with a 2-s drop test and 2-s sinking time) within just 3 min of wicking time. The Polyester/cotton blend fabrics treated with papain enzyme exhibited several noteworthy characteristics, including a significantly reduced susceptibility to fabric pilling (4–5), a limited capacity to attract oily impurities, and a high oil-soil-release capability with a stain removal index of 85%. Additionally, the fabrics showed a one-order-of-magnitude decrease in surface resistivity under normal conditions, with a half-life decay time of 513 s. Observations of the treated fabrics revealed the presence of cracks, grooves, nanostructures, and a high degree of roughness on the surfaces that were treated with papain enzyme. To further evaluate the effects of the lipase enzyme treatment on the fabric properties, several tests were conducted, including Fourier Trasform Infrared spectroscopy (FT-IR), Thermogravimetric Analysis (TGA), Differential scanning calorimetry (DSC), Moisture Regain, Tensile Strength, Stain Repellency, pilling resistance, and Anti-static charge generation.