An autoclavable sensing film was developed for monitoring dissolved CO2. The sensing film, based on fluorescence resonance energy transfer (FRET), consisted of a fluorescent donor, an acceptor, and a quaternary ammonium hydroxide, which were doped in a two-component silicone film. As no aqueous solution was used in the sensing film matrix, the sensing film was unaffected by osmotic pressure. Fluorescence lifetime was selected as the sensing parameter, and measured in frequency domain using phase fluorometry. Upon exposure to 20% CO2-saturated water, a 43 degrees increase in phase angle was observed at 100 MHz. The process was fully reversible when the sensing film was exposed to nitrogen-saturated water. The estimated response and recovery times for 90% signal change were 1 min (for a step change from 0 to 6.7% CO2-saturated water) and 1.5 min (for a step change from 6.7 to 3.3% CO2-saturated water). When used for on-line monitoring of dissolved CO2 produced by a culture of Escherichia coli, the sensing film showed a similar trend to that obtained from off-line measurements using a wet chemistry analyzer.