Surfactant is one of the major consuming auxiliaries in textile processing. The rising demand for petroleum-based surfactants is in focus and it is tremendously utilized to fulfil the need for surfactants in textile industries. These petroleum-based surfactants are one of the major pollutants of textile wastewater. Many attempts have been made to replace this with low toxicity to make the process sustainable. The present investigation works on the same objective to replace the petroleum-based surfactant from desizing by using soapnut extract as a wetting agent. The process was optimised by using a modern statistical technique of Response Surface Methodology [RSM]. The initial designing was conducted using 10 g/l soapnut extract and 2% enzyme for 30 min at 75 °C and found satisfactory results. Additional desizing experiments were performed to optimize the process using RSM with weight loss as the primary outcome. An optimised desizing recipe provided by DOE numerical optimisation, viz., a concentration of 10 g/l soapnut extract and 2% enzyme at 75 °C for 40 min, was performed to validate. The findings demonstrate that optimum weight loss (6.58%) and desirable levels of absorbency (14 s), whiteness (73.52), yellowness (22.84 indices, bending length (2.1 cm), Flexural rigidity (98.13 mg.cm), while minimally affecting tensile strength (10.77). Enzymatic desizing with synthetic or soapnut-extracted wetting agents yields identical results and satisfies performance standards for industrial use. The Sustainable way of enzymatic desizing of cotton with bio-surfactant extracted from soapnut may be the green alternative to synthetic surfactant-based desizing.