The invention discloses a one-bath low-temperature desizing, refining and bleaching pretreatment method of cotton woven fabric. The method comprises the steps of adding 1-5g/ L of bio-enzyme for desizing, 3-8g/ L of bio-enzyme for refining, 2-6g/ L of hydrogen peroxide, 1-4g/ L of a bleach activator, 2-6g/ L of a chelating agent and 0.5-4g/ L of an auxiliary agent into a one-bath system, adjusting the pH value to 6.0-7.5, putting the fabric into a dye bath according to the bath ratio of 1: (10-100), heating up to 50-70 DEG C at the speed of 0.5-2 DEG C/ minute, carrying out heat preservation for 40-120 minutes, finally, washing the fabric with water, and drying or airing. The bio-enzyme for desizing, the bio-enzyme for refining and the bleach activator are added into a pretreatment system of the cotton woven fabric, so that the processes of desizing, refining and bleaching of the cotton woven fabric can be carried out in one bath at a low temperature, the strength damage to the fabric is avoided, the technological process of the dyeing and finishing pretreatment of the cotton woven fabric can be shortened, the energy consumption is reduced, and the production efficiency is increased.