A novel fluorescence microscope configuration for real‐time observation of the Langmuir–Blodgett transfer is presented. It enables the imaging of any portion of the entire monolayer, including on the water subphase, on the substrate, or even on the meniscus. Studies are performed even while the layer is being deposited. Two sets of optical components, one for the visible and one for the ultraviolet spectrum, were implemented. Studies on a monolayer of dimyristoyl‐L‐α‐phosphatidylethanolamine (DMPE) reveal domain growth and nucleation phenomena at the boundary between water‐supported and deposited film. The performance of the instrument in the UV is demonstrated with pentadecanoic acid containing a pyrene probe.

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