Scientific interest in the self-assembly of collagen composite films has been increasing for their potential application in constructing bioactive materials. Here we report a highly stable and cytocompatible collagen/alginate (COL/ALG) ultrathin film, which was linearly fabricated via a layer-by-layer self-assembled technique. The variation in morphology and thickness of the films in air and in solutions with different pH and ion values were tested by atomic force microscopy. Results showed that the solutions with high pH values or solutions that contained electrolytes would disintegrate the film, while films with that were cross-linked for a long time prevented the dissolution and contributed to stability maintenance of the films. Interestingly, the COL/ALG coating not only improved the adhesion and proliferation of the human periodontal ligament cells, but also modified the morphology and migration of cells on the surface of glass and poly-L-lactic acid (PLA) electrospun scaffolds. In conclusion, the COL/ALG ultrathin films were highly stable and cytocompatible and could be easily fabricated by the cost-effective self-assembled technique presented. The findings of this study have the potential to play an important role in the surface modification of biomaterials.