X-ray reflectometry (XR) was carried out on amphiphilic diblock copolymer poly( a-methylstyrene)bloch-poly(decyl 4-vinylpyridine) monolayers on a water surface and on a glass plate. From XR data, the layer thickness and surface and interface roughness were determined. For comparison, atomic force microscopy (AFM) was carried out on the samples deposited on solid substrates (glass and mica). From XR data, it was quantitatively clarified that the monolayer became thicker and rougher by the deposition on solid substrates than on the water surface. AFM revealed aggregates formed on the solid substrates. The structural difference between the samples on a water surface and on solid substrates may be due to the preparation procedure of the sample on solid substrates. These findings indicate that the correct and precise information far the structure of the monolayer on water surface can be evaluated only by in situ experiments such as XR.