Abstract: A series of P(DMA-co-HEA) copolymers with varying structural unit ratios of N, N-dimethylacrylamide (DMA) and hydroxyethyl acrylamide (HEA) were synthesized via solution polymerization. The hydrogen-bonding interactions in the polymer solutions and the effects of mass concentration, temperature, and shear rate on the rheological properties of the polymer solutions were systematically investigated. Concurrently, The copolymers were employed as sieving matrices in capillary electrophoresis applications to assess the impact of HEA content on the sieving performance of deoxyribonucleic acid(DNA) fragments. Rheological results revealed that, across different mass concentrations and temperatures, the thickening ability of P(DMA-co-HEA) reached its maximum at a structural unit molar ratio of n_(DMA)/n_(HEA) was 29.92. This indicates that an appropriate amount of HEA units can significantly enhance the shear resistance and thermal stability of the copolymer. stability of the polymer solutions through hydrogen bonding interactions. Furthermore, the evaluation of viscoelastic behavior demonstrated that a stable, weak hydrogen-bonded physical network is formed within the system when n_(DMA)/n_(HEA) ranges from 37.28 to 19.44. DNA fragment analysis further confirmed that the proper incorporation of HEA facilitates the enhancement of DNA sieving capability. Notably, when n_(DMA)/n_(HEA) was 29.92, the sieving matrix exhibited optimal separation performance, minimized peak attenuation, and excellent analytical reproducibility.