Silk fibroin (SF) is a promising candidate for a variety of application in the fields of tissue engineering, drug delivery, and biomedical optics. Recent research has already begun to explore the construction of nano- and micropatterned SF films under ambient environment. The structure and biocompatibility of SF are dependent on SF state (solution or solid) and the method of drying the SF solution to prepare various biomaterials such as films, sponges, and fibers. Therefore, it is important to explore the construction of SF nano- and micropatterns under aqueous solution. This paper reports a novel application of atomic force microscopy (AFM) under liquid for direct deposition of the relatively hydrophobic protein SF onto hydrophilic mica. We demonstrate that the AFM tip, in either the contact or the tapping mode, can fabricate SF micropatterns on mica with controlled surface topography. We show that the deposition process requires a mechanical force-induced SF sol-gel transition followed by a transfer to the mica surface at the tip-surface contact, and the efficiency of this process depends on not only AFM operation mode but also the SF bulk concentration, the SF amount on mica, and the AFM tip spring constant.