In our previous study, semi-transparent and flexible biodegradable films were successfully prepared from myofibrillar proteins of blue marlin meat. The objective of this study was to elucidate the mechanism of film formation. Effect of pH on the amount of surface hydrophobicity and surface SH group in the film-forming solution, solubility of film proteins in various protein denaturant solutions, SDS-PAGE, and addition effect of protein denaturants in the film-forming solutions were investigated. At first, the necessity of unfolding of protein molecules was revealed for the formation of self-standing films. Secondly, the important chemical bondings involved in the protein-protein interactions were confirmed to be hydrophobic interaction for acidic condition, hydrogen bondings for neutral condition, and hydrophobic interaction and disulfide bondings for alkaline condition.