Reducing input of chemical fertiliser to cotton fields has the benefits of reduced costs, increased crop production efficiency, and positive environmental impact. We investigated the impacts of stalk management and fertiliser application on the morphology, physiological activity and nutrient use efficiency of cotton roots in arid areas when grown under drip irrigation with plastic film mulching. This 9-year field experiment involved a split-plot design with two stalk management treatments (removed or mixed with soil) as main plots and four fertiliser treatments (no fertiliser, NPK fertiliser, poultry manure, combined NPK fertiliser plus poultry manure) as subplots. Fertilisation increased root tissue mass density, specific surface area, triphenyl tetrazolium chloride reducing capacity, and glutamine synthetase, but decreased specific root length. Straw return and fertilisation increased soil available N, P and K. Nutrient use efficiencies of N, P and K in roots were sensitive to fertilisation treatment, whereas nutrient use efficiencies in total dry matter were sensitive to straw return treatment. Fertilisation increased the root:shoot ratio, root biomass, fine:coarse root mass ratio, and seed cotton yield. Combined application of NPK fertiliser and poultry manure can prompt the formation of fine roots, which maximised the contact area between poultry manure and roots, thus increasing the chance of the root acquiring mineral nutrients. Therefore, organic matter promotes the absorption of mineral nutrients by roots, thereby improving the efficiency of fertiliser utilisation.