The conformational rearrangement of poly(methyl methacrylate) (PMMA) chains confined in a two-dimensional monolayer to a three-dimensional random coil was studied by the shape and segment density analysis of an individual PMMA chain, and was directly observed by scanning near-field optical microscopy. A perylene-labeled PMMA chain was dispersed in the unlabeled PMMA monolayer, after which it was placed on a thick film of unlabeled PMMA and annealed above the glass transition temperature. Although the polymer chain of the monolayer penetrated into the bulk substrate with thermal activation, the radius of gyration in the lateral direction was maintained throughout the annealing process. The diffusion rate in the depth direction was much faster than that of the translational diffusion of the entire polymer chains, suggesting that an unstable twodimensional conformation of the polymer chain relaxed into a three-dimensional one. This indicates that the polymer chains confined in a monolayer interlaced with the surrounding chains and expanded mainly toward the depth direction in the relaxation process, while maintaining the dimensions of the averaged radius of gyration in the lateral direction.