Mechanical properties of intestinal organoids are crucial for intestinal development, homeostatic renewal, and pathogenesis. However, characterizing these properties remains challenging. Here, we developed a microinjection-based method to quantify the growth time-dependent nonlinear elasticity of intestinal organoids. With aid of the neo-Hookean hyperelastic constitutive model, we discovered that the global elastic modulus of intestinal organoids increased linearly during the early stages of culture, followed by a sharp rise, indicating a time-dependent nonlinear hardening behaviour during growth. The global modulus of intestinal organoids was found to correlate with the cell phenotype ratio, revealing a significant relationship between mechanical properties and biological phenotypes. Furthermore, we developed a biomechanical model on the basis of the unsteady Bernoulli equation to quantitatively explore the global mechanical responses of intestinal organoids, which showed good agreement with the experimental data. The work not only elucidated the mechanical response and modulus characteristics of small intestinal organoids from a biomechanical perspective, but also presented a new microinjection-based methodology for quantifying the mechanical properties of organoids, offering significant potential for various organoid-related applications. STATEMENT OF SIGNIFICANCE: Mechanical properties of intestinal organoids are essential for intestinal development, homeostatic renewal, and pathogenesis. However, how to quantitatively characterize their global mechanical properties remains challenging. Here, we developed a new microinjection-based experimental platform to quantify spatiotemporal dynamics of mechanical responses and global elasticity of intestinal organoids. Unlike traditional nanoindentation methods, the proposed characterization technique can quantitatively measure the global mechanical properties of organoids, which is crucial for detecting the inherent relationship between the global mechanical properties and the biological phenotypes of organoids. Likewise, it established a methodological foundation for revealing the mechanobiological characteristics associated with the growth and development of various organoids. This can enhance our understanding of mechanobiological mechanisms of organoids and is beneficial for various organoid-related applications.