In situ ultraviolet photoemission spectroscopy was used during the pentacene layer growth on a Ru film on a Ti adhesion layer (Ru/Ti) to measure the energy barrier in the metal–semiconductor contact. A Ru surface was treated with a 4-fluorothiophenol and 4-methylbenzenethiol self-assembled monolayer to improve the interface properties between the metal electrode and the pentacene layer. The chemical bonding state of the self-assembled monolayer (SAM) on the surface of Ru/Ti film was confirmed using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The Ru/Ti film showed excellent electrical properties as source and drain electrodes of the pentacene thin film transistor (TFT) owing to the high work function of 4.64 eV and low resistivity of ∼160 μΩ cm. It was confirmed that 4-fluorothiophenol SAM treatment on Ru/Ti film increased the work function of the metal to 5.1 eV and the grain size of the pentacene layer grown on SAM was also increased. With the reduction in the barrier height, the pentacene TFTs with 4-fluorothiophenol treated Ru/Ti film showed a higher mobility of 1.03 cm2/Vs and on-off ratio of 5 × 106 than a pentacene TFT with bare Ru/Ti (μ: 0.205 cm2/Vs, on/off ratio: 106) or bare Au/Ti (μ: 0.275 cm2/Vs, on/off ratio: 106) of the same structure.