A n-type organic semiconductor, N,N'-didodecyl-3,4,9,10-perylene tetracarboxylic diimide (PTCDI-C12H25), film crystallizes on various temperature substrates to act as active layer of organic thin-film transistors (OTFTs). The nuclear magnetic resonance 1-D hydrogen spectroscopy (1H-NMR) was used to analyze molecular structure of the PTCDI-C 12H25 which was synthesized by us. When the transistors was fabricated on room temperature substrate, the field-effect mobility of the device was about half of that of the transistor fabricated on high temperature substrate. Furthermore, x-ray diffraction analysis gave an account that the higher temperature substrate provided better epitaxial environment for PTCDI-C12H25 thin film growth. The grain size of PTCDI-C12H25 thin films deposited on room temperature and 100 °C substrates were 24.8 and 35.6 nm, respectively, which analyzed by paracrystalline theory. We base that larger grain results in better carrier transport in organic films on the grain boundary model which assumed that the boundary of grain will trap charge carriers when they pass through these grains, and the quality differences of microstructural within a grain can be neglected. The PTCDI-C12H25 based OTFTs were achieved high performances, such as field-effect mobility of 0.10 cm2V -1s-1, threshold voltage 14.5 V, on-off ratio 3.01 × 106 and subthreshold swing 1.24 V/dec, when the active layer was grown under substrate temperature of 100 °C. Additionally, contact resistance analysis reveals that PTCDI-C12H25 thin film on 100 °C substrate has better interface between organic thin film and electrode.