Abstract: Objective To monitor the 3-dimensional (3D) morphological changes of C6/36 cells during dengue virus (DENV) infection using a live-cell imaging technique based on digital holographic microscopy and provide clues for better understanding the mechanisms of DENV infection. Methods C6/36 cells were seeded in 6-well plates to determine the optimal imaging density under a holographic cell imager, and the morphological changes of the cells were recorded in response to a culture temperature change from 28 ℃ to 37 ℃. C6/36 cells were infected with 4 DENV strains with different serotypes at 28 ℃ and incubated at 37 ℃ for 24 h, and the 3D holograms and relevant morphological parameters were recorded at different time points using HoloMonitor M4 holographic cell imaging and analysis system. Results The holograms of C6/36 cells inoculated at the optimal density for imaging (4 × 105 per well) showed unified 3D morphologies of the single cells with minimal dispersions in the cell area, thickness and volume (P<0.05), which did not undergo obvious changes when the cells were incubated at 37 ℃ for 24 h (P>0.05). The cell area and volume of the cells infected with the 4 DENV strains all increased and the cell thickness was reduced during incubation. Among the 4 strains, DENV-1 and DENV-2 caused reduced cell thickness while DENV-3 and DENV-4 increased the cell thickness, and the pattern and degree of such changes differ among the 4 strains. Conclusions Digital holographic microscopy allows monitoring of the complex morphological changes of cells during DENV infection. The 4 DENV strains with different serotypes causes characteristic cell damages during infection.