Inspired by the hierarchical structure of nacre, lots of functional nanocomposite films have been fabricated through finely tuning the composition and structure. To broaden their application in biological and biomedical research, it is highly demanded to develop a new strategy to fabricate nanocomposite films that are both biocompatible and functional. Herein, hyperbranched poly(amido amine)s (HPAMAMs) have been assembled with clay nanosheets. The obtained nanocomposite films show good mechanical performance and fluorescence properties. Analysis of the structure of the nanocomposite films indicates that the HPAMAMs and the clay nanosheets are stacked together in an orderly manner. The clay nanosheets align in the direction parallel to the film surface. Because of the inherent biocompatibility and autofluorescence property of the HPAMAMs, such composite films are thought to be very promising in applications requiring biocompatibility, fluorescence, and good mechanical performance. The method should provide a new route to the fabrication of functional nacrelike composite materials.