Culture fluorescence measurement is an indirect and non-invasive method of biomass estimation to assess the metabolic state of the microorganism in a fermentation process. In the present investigation, NAD(P)H fluorescence has been used for on-line in situ characterization of metabolic changes occurring during different phases of batch cultivation of Azohydromonas australica in growth associated poly(3-hydroxybutyrate) or PHB production. A linear correlation between biomass concentration and net NAD(P)H fluorescence was obtained during early log phase (3-12 h) and late log phase (24-39 h) of PHB fermentation. After 12 h (mid log phase) cultivation PHB accumulation shot up and a drop in culture fluorescence was observed which synchronously exhibited continuous utilization of NAD(P)H for the synthesis of biomass and PHB formation simultaneously. A decrease in the observed net fluorescence value was observed again towards the end of fermentation (at 39 h) which corresponded very well with the culture starvation and substrate depletion towards the end of cultivation inside the bioreactor. It was therefore concluded that NAD(P)H fluorescence measurements could be used for indication of the time of fresh nutrient (substrate) feed during substrate limitation to further enhance the PHB production.