Quantitative characterization of the mechanical properties of a polystyrene (PS) monolayer colloidal crystal (MCC) annealed with solvent vapor has been performed for the first time by means of atomic force microscopy nanoindentation. The results showed that both the compressive and bending elastic modulus of PS MCC increased with the prolongation of annealing time from initial to 13 min. When the annealing time reached 15 min or even more, the PS MCC almost deformed to a planar film, and the elastic modulus of the PS MCC presented a drastic increase. These results provide a basis for tailoring the mechanical properties of a polymer colloidal monolayer via solvent vapor annealing. Such self-supported and high-mechanical-strength colloidal monolayers can be transferred to other surfaces for potential and promising applications in the bottom-up fabrication of highly ordered nanostructured materials such as nano dot arrays, photonic crystals, and many others.