Objective：To investigate the female vaginal Lactobacillus crispatus biofilm by using confocal laser scanning microscopy (CLSM)，thus revealing the formation of biofilm. Methods:The cover slide biofilm culture approach in vitro was employed for induction of the vaginal Lactobacillus crispatus biofilm formation．Following the culture for 2， 4， 8， 12， 16， 20, 24， 48, 72, 96 and 120 hours, the cover slide was removed for subsequent staining with the fluoresce in isothiocyanate-conjugated concanavalin A(FITC-ConA) and propidium (PI).This was followed by determination of the formation and characteristics of the vaginal Lactobacillus crispatus biofilm by using CLSM. Results:The CLSM images of biofilm formation at different time points were captured, suggesting that the vaginal Lactobacillus crispatus adhesion occurred at h 4， which was in reversible attachment, then more and more Lactobacillus crispatus aggregated at h 8  to h 20, which was in irreversible attachment．Lactobacillus crispatus clustered at h 20, with early development of biofilm architecture．Then the biofilm with extracellular matrix around the bacteria was set up at h 24，with gradual matureation at h 24 to h 48．The biofilm dispersed at h 72. The biofilm density of cultivating for 20 hours was 42.7×10-3±6.8×10-3，and for 24 hours increased to 102.5×10-3±23.1×10-3, suggesting a significant difference, P<0.05. This meant that mature biofilm was formed at h 24. Conclusion:The vaginal Lactobacillus crispatus is able to form typical biofilm with distinct developmental phases and architecture characteristics．Mature biofilm is formed at h 24 to h 48, then the biofilm begins to disperse.