Large-scale close-packed two-dimensional (2D) colloidal crystal with high coverage is indispensable for various promising applications. The Langmuir-Blodgett (LB) method is a powerful technique to prepare 2D colloidal crystals. However, the self-assembly and movement of microspheres during the whole LB process are less analyzed. In this study, we clarify the crucial impact of hydrophilicity of the microspheres on their self-assembly in the LB process and on the properties of the prepared 2D colloidal crystals. The characteristic surface pressure-area isotherms of the microspheres have been analyzed and adjusted by only counting the quantity of the microspheres on the water surface, which leads to more accurate results. The critical surface pressures for hydrophilic and hydrophobic microspheres are about 61 and 46 mN/m, respectively. The decrease of the surface hydrophilicity of microspheres facilitates their self-assembly on the water surface, which further leads to higher coverage and less defects of the 2D colloidal crystals. A coverage of as high as 97% was obtained using hydrophobic microspheres. Entropy and intersphere capillary forces drive the self-assembly and transportation of the microspheres, respectively. Caused by the diffraction of visible light, opposite contrasts at local adjacent regions on the surface of the 2D colloidal crystals have been observed. The understanding of self-assembly of the microspheres during the LB process paves the way to fabricate the high-quality 2D colloidal crystals for various applications such as photonic papers and inks, stealth materials, biomimetic coatings, and related nanostructures.