The surface-pressure, surface-area isotherms of pure bovine serum albumin (BSA) and mixed poly-(D,L-lactide) (PLA50)/BSA monolayers spread from a water-in-oil emulsion at the air/water interface were obtained and compared with those of monolayers of the same constituents obtained by usual methods. In order to obtain additional information about the film organization, a rheological approach based on Maxwell’s model and AFM imaging were used. The results show that the two constituents are better mixed in the monolayers formed by spreading an oil-in-water emulsion, while a greater segregation exists in the monolayers formed by successive spreading. The presence of BSA aggregates was observed in the monolayers formed from emulsion. This could have some consequences on the preparation and the properties of the PLA50 particles obtained by an emulsion technique.