This study investigated polyethylenimines (PEIs) with varied architectures as low toxicity and efficient catalysts for aqueous isomerization of glucose to fructose. Under the investigated reaction conditions, the studied PEIs achieved 33–36% maximum fructose yields with 66–77% selectivity at 110–120 °C, with the branched PEI generally outperforming the linear and comb PEIs. Moreover, with 1 wt % sodium chloride, the PEIs achieved 35–38% maximum fructose yields with 61–72% selectivity at 110 °C. After modification through room temperature cross-linking, the branched PEI was transformed into a recyclable heterogeneous catalyst with similar isomerization performance (30% fructose yield and 78% fructose selectivity at 120 °C) to the homogeneous PEIs. Remarkably, in the presence of neutral salts, the heterogeneous PEI achieved an approximately 41% fructose yield with 72–78% selectivity at 110 °C and showed an excellent reusability.