This study revealed the influence of phosphorylation-dimethylammonium chloride acyloxylation (PDACA) on the desizability, film properties, paste stability, and adhesion of biological starch macromolecules. A new starch-based sizing agent, phosphorylated-dimethylammonium chloride acyloxylated starch (PDACAS), was synthesized with degrees of substitution (DS) ranging from 0.033 to 0.065. Compared to control phosphorylated-quaternized starch (PQS, 87.4 %), the desizing efficiency of cotton yarns sized with PDACAS was ~94 %, exceeding the industrial minimum requirement of 90 %. The PDACAS film tensile properties were as follows: elongation at break of 3.31 %–3.78 %, bending endurance of 1131–1537 cycles, and tensile strength of 35.83–28.31 MPa, compared with those of acid-thinned starch (ATS) film (2.74 %, 957 cycles, and 38.12 MPa). The PDACAS had paste stability of ~92 %, compared with 83.3 % for ATS. The bonding forces (an indicator of adhesion to fibers) ranged from 107.1 N to 125.3 N for cotton roving, and 128.3 N to 148.7 N for polyester/cotton roving, which were significantly better than those of ATS (95 N for cotton and 117.9 N for polyester/cotton roving). Overall, PDACA treatment effectively avoided the adverse effect of high DS quaternization on the desizability of PQS and imparted good film properties, paste stability, and adhesion to starch.