In this paper, we developed a low-cost and easy-to-fabricate hydrogel-based localized surface plasmon resonance (LSPR) substrate for sensitive, consistent, specific, and time-lapse detection of hydrogen peroxide (H2O2) secreted from living cells in cell culture medium. The uniform and reproducible LSPR substrate was made from agarose, gold nanorods (AuNRs), horseradish peroxidase (HRP), and 3-amino-9-ethylcarbazole (AEC). In H2O2 sensing, insoluble precipitates were generated in the gel film by an HRP-catalyzed reaction in the presence H2O2, and the precipitates near the AuNRs caused a LSPR shift of the substrate, which could be used for H2O2 quantification. Using the developed substrate, we showed that a H2O2 concentration as low as 0.5 μM could be detected in complete cell culture medium within 30 min with good reproducibility. In addition, we demonstrated time-lapse detection of H2O2 secreted from living cells after the cells were stimulated by ascorbic acid. The results indicated that the agarose gel of the substrate provided the filtration capability and the improved stability of the AuNRs allowing detection of H2O2 in cell culture medium without tedious sample preparation procedures. Moreover, the HRP-catalyzed reaction-based LSPR biosensing offered good selectivity for H2O2 detection and possessed great potential for various in vitro cell studies.