The advent of atomic force microscopy (AFM) provides a powerful tool for probing the physical properties of native biological samples with unprecedented spatiotemporal resolution at the micro/nanoscale, significantly complementing traditional biochemical assays. In recent years, multiparametric imaging AFM, which is able to simultaneously obtain the topographical features and multiple mechanical properties (e.g., Young’s modulus, adhesion, and deformation) of biological samples, emerges as a new method for investigating the correlations between structure, mechanics and functions of biological systems. The biomedical applications of multiparametric imaging AFM have yielded considerable novel insights into cellular/molecular activities and the underlying mechanisms guiding pathological processes. In this paper, based on our own research in single-cell detection using AFM, the principle of multiparametric imaging AFM is presented and the progress in utilizing multiparametric imaging AFM to probe cellular and molecular mechanics is summarized. The challenges and future directions are also discussed.