We herein report a method for the preparation of a glass microchannel capable of forming multiple emulsion droplets (i.e., water-in-oil-in-water and oil-in-waterin-oil) by locally controlling the wettability of the glass microchannel. Production of multiple emulsion droplets using a glass microchannel requires partial control of its wettability using a method that consists of two steps: (1) hydrophobization of a whole glass microchannel by filling the microchannel with octadecyltrichlorosilane (OTS) solution, and (2) local hydrophilization of the OTS-treated glass microchannel by exposure to ultraviolet light through a mask. However, conditions for the preparation of OTS-SAMs for controlling microchannel wettability and subsequent multiple emulsion droplet formation have not yet been reported. In this study, we investigated the conditions required to form multiple emulsion droplets and demonstrated formation of multiple emulsion droplets using a treated glass microchannel with multiple junctions. The glass microchannel prepared according to this method was able to form various aqueous and organic droplets due to its resistance to swelling.