The transfer free energy (TFE) of apomyoglobin (AMb) from pure water into aqueous solution with trimethylamine N-oxide (TMAO) was investigated by all-atom molecular dynamics (MD) simulation combined with the Kirkwood-Buff (KB) integral method. The simulated TFE and the preferential interaction parameter correlated favorably with experimental values. In addition, the time-resolved KB integral revealed that a significant fluctuation in the TFE arose from the alteration in TMAO solvation around AMb. Furthermore, spatial decomposition of the KB integrals revealed how the local elements of the TFE are spatially distributed around AMb. These results revealed the spatio-temporal characteristics of the protein TFE into the molecular crowding condition with TMAO.