Objective The ability of the single-species and multi-species biofilm formation on the artificial root surface by Streptococcus sanguis（S.sanguis）, Fusobacterium nucleatum（F.nucleatum） and Porphyromonas gingivalis （P.gingivalis） was observed in order to establish an in vitro subgingival plaque biofilm model. Methods S.sanguis, F.nucleatum and P.gingivalis were cultured, and collagen-treated hydroxyapatite was prepared as the artificial root surface. The formation of the single-species, dual-species and multi-species biofilms of S.sanguis, F.nucleatum and P.gingivalis on the artificial root surface were detected at 24, 48 and 72 h by scanning electron microscopy. Results S.sanguis biofilm was formed on the artificial root surface at 24 h, and showed a three-dimensional structure. The biofilm looked more mature at 48 and 72 h. P.gingivalis biofilm was formed at 48 h, and showed a three-dimensional structure. The density of P.gingivalis in the biofilm declined at 72 h. P.gingivalis and F.nucleatum were able to form a complete dual-species biofilm with full three-dimensional structure at 24 h. The dualspecies biofilm of P.gingivalis and F.nucleatum lost the complete structure at 48 and 72 h, in which a significant reduction in the number of two types of bacteria was observed. S.sanguis, F.nucleatum and P.gingivalis formed a three-dimensional structure multi-species biofilm at 24 h. P.gingivalis shared lower proportion in the number of the three bacteria. The biofilm became more mature at 48 h. The proportion of P.gingivalis increased. At 72 h the biofilm remained a complete structure with a decline of the number of the three bacteria. Conclusion The single -species（S.sanguis or P.gingivalis）, dual-species（F.nucleatum and P.gingivalis） and multi-species（S.sanguis, F.nucleatum and P.gingivalis） biofilms were formed at 24, 48 and 72 h in this study. 24 hours may be an optimal period for establishing the biofilmmodel of periodontal bacteria for the further study.
