Abstract: OBJECTIVE To compare the biofilm(BF) formation ability of Aspergillus fumigatus between flow chamber system and static system in vive, to find out the best external study model, so as to provide technical support to external study. METHODS There were two in vitro biofilm models of A.fumigatus established: flow chamber system model and static model, which were stained the extracellular matrix(ECM) with FITC-Canavalia ensiformis(FITC-ConA) at different time points, and their three-dimensional (3D) biofilm morphology with laser scanning confocal microscope (CLSM) were observed. The 3D image stacks were under quantitatively analysis with COMSTAT software to compare the growth index (e.g. biomass and average thickness) between two groups. RESULTS Within each model, biomass, average thickness, substrate coverage, and the average diffusion distance increased according to time period, while roughness coefficient, surface area to biomass ratio decreases decreased with time; between groups, biomass, average thickness, substratum coverage and average diffusion of BF formed in static model were significantly higher than that of continuous flow system under the same time point (P<0.05), while roughness coefficient, surface to biovolume ratio were significantly lower (P<0.05), but the following one had not. CONCLUSION A. fumigatus can form biofilm stably in flow system, but the biofilm under static system can secrete more ECM and form a thicker biofilm, which is more appropriate for A.fumigatus biofilm studies in vitro.