Objective Detecting the detailed behaviors of single viruses is essential for uncovering the underlying mechanisms guiding virus life cycle, which significantly benefits developing therapeutic methods against viral infection. The advent of atomic force microscopy (AFM) provides a novel powerful tool to characterize the structures and mechanical properties of single viruses with unprecedented spatial resolution, and applications of AFM in single-virus assay have contributed much to the field of physical virology. Nevertheless, the mechanical cues of single native viruses during viral activities are still not fully understood, and particularly studies of utilizing multiparametric AFM imaging to investigate the behaviors of single viruses are still scarce. Here, multiparametric AFM imaging was combined with AFM indentation assay to investigate the structural and mechanical dynamics of single native virus particles in response to chemical stimuli under aqueous conditions.Methods The poly-L-lysine was used to coat the coverslips to attach lentivirus particles onto the coverslips, and then the virus particles were probed by AFM in pure water. Single virus particles were imaged at the peak force tapping (PFT)-based multiparametric AFM imaging mode, in which the topographical images and mechanical maps of the virus particles were obtained simultaneously. Under the guidance of AFM’s topographical imaging, the AFM probe was moved to the central area of the virus particle to perform indentation assay for measuring the mechanical properties of the virus. The alcohol solution (75%) was used as an example of chemical stimulus to treat virus particles, after which the structural and mechanical changes of individual virus particles were revealed by AFM.Results The structures and mechanical properties of single virus particles could be well characterized by AFM under aqueous conditions, and the virus particles exhibited different elastic and adhesive properties in air and in liquid. After the treatment of alcohol, the shape of virus particles became irregular, and the virus particles became stiffer as well as less deformable.Conclusion The research provides a novel way to investigate the structures and nanomechanical properties of single native viruses in liquids based on AFM, which will have general implications for the field of virology.