H2O2 generation by glucose oxidase (GOx)-catalysed oxidation of glucose was used to supply H2O2 in the horseradish peroxidase (HRP)-catalysed hydrogelation of aqueous solutions of polymers bearing phenolic hydroxyl (PH) moieties. The feasibility of the glucose-triggered hydrogelation was evaluated using an alginate derivative bearing PH moieties (Alg-PH). The initial rate of cross-link formation between the PH moieties and the gelation time of the Alg-PH aqueous solution increased and decreased, respectively, with increasing GOx and glucose concentrations. Hydrogels with comparable cross-link densities and gelation times to those formed using a conventional direct H2O2 addition system could be obtained by altering these parameters. The mechanical properties of the resultant hydrogels were also controllable. The hydrogel obtained from a 1.1% (w/v) Alg-PH solution at 1 u mL−1 HRP, 0.8 u mL−1 GOx, and 4.6 mM glucose showed repulsion forces toward compression more than four times those shown by hydrogels obtained with 1 u mL−1 HRP and 4.6 mM H2O2. These results indicate that the glucose-triggered hydrogelation system has potential for a wide range of biomedical applications such as fabricating substrates for tissue engineering, carriers for drug and protein delivery systems, and wound dressings.