This paper describes an application of fluorescence spectroscopy for in-situ studies of the structure and properties of monolayer films, especially of pre-formed polymer films at the air-water interface. Poly(vinyl alkanal acetal)s were synthesized and a part of the side chains were labeled with pyrene chromophores. The fluorescence intensity and lifetime of pyrenes are strongly quenched by oxygen in the air. The surface film sensitively responds to alternation of the surroundings. This behavior can be used as a probing technique for the packing state of the monolayer. The excimer formation of pyrenes reveals the dynamic behavior of the monolayer. Time-resolved measurements show a clear rise in excimer emission, i.e. the excimer is formed by microscopic diffusion of polymer segments. These results indicate that the monolayer maintains its mobility even in the condensed phase. Fluorescence spectroscopy provides molecular information and a valuable technique for studying monolayers in situ.