The invention relates to a pre-processing, polishing and dyeing one-bath process for cotton knitted fabric; the pre-processing, polishing and dyeing one-bath process comprises the following steps of: processing the cotton knitted fabric by adopting pectinase, neutral cellulase, a refined penetrating agent and a buffer solution, removing a part of or all pectin, wax, oil stains, ash contents and other impurities affecting the dyeing performance on the knitted fabric, endowing the fabric with good wettability and then carrying out a conventional reactive dye dyeing process. As a method of combining biological enzyme and a chemical assistant is adopted, the fabric is deeply colored and has good hand feeling. The pre-processing, polishing and dyeing one-bath process provided invention has the beneficial effects that as the three procedures, i.e. processing, polishing and dyeing the cotton knitted fabric are combined into one bath, the processing procedure is reduced, the production efficiency is improved, the high-temperature treatment is avoided, and the wastewater discharge quantity is reduced; and moreover, the requirements of energy conservation and emission reduction in the dyeing industry are satisfied.