Biofilms occur in natural and engineered water systems. Biofouling in technical processes lowers the water quality and increases the frictional resistance in tubes. In wastewater treatment plants, biofilms are used for removal of organic an inorganic pollutants. For improvement of antifouling strategies and for process optimization in wastewater treatments plants, an analytical technique for online monitoring of biofilms is needed. In this article, a new setup for in situ monitoring of biofilms by photoacoustic spectroscopy is presented. To produce a biofilm, a mixture of microorganisms was grown in a nutrient solution inside a tube reactor. The content of the tube reactor was pumped through a flow channel, and biofilms were generated at the inner surfaces. Three photoacoustic sensor heads were integrated at different positions into the base plate of the flow channel. By photoacoustic spectroscopy, growth, thickness, and detachment of biofilms can be monitored on-line and nondestructively. Experiments presented in this article showed that the flow conditions influence the structure and thickness of biofilms. By changing the pH value, electrostatic interactions inside the biofilm matrix were influenced, and the subsequent detachment processes were observed online. The interaction of iron(III) oxide particles with biofilms led to particle adsorption on the outer and inner surfaces of the biofilm. Afterwards, biofilm flocs were sloughed off from the base biofilm.