Microenvironmental changes in the chemical surrounding of bacterial cells might have a profound impact on the ecology of biofilms. However, quantifying total amount of picomoles of analyte from a miniscule number of bacteria is an analytical challenge. Here we provide a novel microliter volume hydrogel based electrochemical cell platform suitable of coulometrically measuring hydrogen peroxide (H2O2) produced by less than 100 cells of Streptococcus sanguinis, a relevant member of the healthy oral microbiome. A morpholine moiety was incorporated into the polymer structure of the hydrogel to create a controlled microenvironment at biological pH. We calculated the buffering capacity of this hydrogel as 0.257 ± 0.135 over the pH range of 7.2-6.2 by using a novel method designed for buffering hydrogels. The H2O2 sensors coated in microliter volume of buffering hydrogel showed no change in sensitivity within the pH range of 7.0-3.0, allowing for H2O2 measurements of S. sanguinis independent of any acid they produce. The novel platform was able to measure down to 22.7 ± 3.5 pmol H2O2 produced by less than 100 bacterial cells, which would otherwise not be attainable in large solution-based assays. These findings indicate that this is a suitable platform for quantifying metabolites from sub-milligram biological samples and may even be suitable for direct analysis of raw biofilms samples with little to no sample pretreatment.