Microbial respiration-based microbiosensors used for quantification of available dissolved organic carbon (ADOC) instantaneously respired by microorganisms are described. The sensing membranes contained aerobic seawater microorganisms immobilized in a polyurethane hydrogel. Molecular investigations revealed that the bacterial strain used was most closely related to Staphylococcus warneri. This strain was characterized by low substrate selectivity, which was reflected in the response to various mono- and disaccharides, short-chain fatty acids, and amino acids, as determined using Biolog microplates. Specific emphasis was placed on critically assessing biosensor functioning that was affected by preconditioning of the selected bacterial strain, chemical and geometric properties of the sensing membrane (e.g., composition, permeability, and thickness), and the distribution, biomass, and physiological state of immobilized cells, as well as the exposure conditions (e.g., temperature and nutrient supply). The sensors revealed that there was a linear response up to a glucose concentration of 500 μM depending on the type, characteristics, and recent history of the sensors. The detection limit of the sensors was equivalent to about 6 to 10 μM glucose. The 90% response time ranged from 1 to 5 min. Generally, the response of the biosensors became weaker with time. The shelf lives of individual sensors were up to 2 weeks. Measurements based on optical ADOC microbiosensors revealed that in photoautotrophically dominated sandy coastal sediments, the pool sizes and turnover of ADOC were regulated by the photosynthetic activity of benthic microalgae and microbial aerobic respiration. A large increase in ADOC production was observed shortly after the microphytobenthic primary production reached the maximum value at midday, whereas ADOC was consumed by microbial respiration during the night.