Combining ultraviolet photoemission spectroscopy (UPS), X-ray photoemission spectroscopy and atomic force microscopy (AFM), we perform a systematic investigation on the correlation of energy level alignment, film growth and molecular orientation of 2, 7-dioctyl[1]benzothieno-[3, 2-b][1]benzothiophene (C8-BTBT) on Ni(100). The molecules lie down at the first layer and are partly devulcanized by the substrate. Chemical adsorption of reaction products of sulfur atoms on the Ni substrate and the evaporation of the hydrocarbon products into vacuum make the C/S ratio as low as 11.5 : 1 in the XPS of the initially deposited C8-BTBT film of 1-4 thickness, far less than the stoichiometric of 15 : 1. With the thickness increasing from 4 to 8 , there are sharp downward shifts of Evac, HOMO and core levels of C 1s, S 2p, and a sharp increase of C/S ratio, which can be ascribed to the change of molecular orientations from lying down at 4 to standing up at 8 . From 8 onward, the C/S ratio increases steadily till it reaches 15 : 1. The energy levels show relatively less changes when the thickness increases from 8 to 32 . When the thickness increases over 32 , the energy band starts bending downward apparently because of the charging effect during the photoelectron emission processes. The poor conductivity along the standing alkyl chain of C8-is the main cause for the charging. The standing up configurations of the C8-BTBT molecules are confirmed by the AFM investigation in which the heights of the upper layers of C8-BTBT are around 30 , close to the length of the long c-axis. AFM image also indicates that the molecules tend to grow into islands for larger thickness, which is consistent with the slower decrease of the (I/I0) of Ni 2p3/2 with the C8-BTBT film thickness. Our results suggest that a buffer layer be inserted between Ni and C8-BTBT and the thickness of the C8-BTBT film be controlled as thin as possible in related devices.