Thiophene compounds are the main concern of petroleum desulfurization, and their chemical composition and molecular configuration have critical impacts on thermodynamic and kinetic processes. In this work, atmospheric pressure chemical ionization (APCI) was employed for effective ionization of thiophene compounds in petroleum with complex matrix, in which carbon disulfide was used for generating predominant [M]+• ions without the need of derivatization as for electrospray ionization. APCI coupled with ultrahigh-resolution mass spectrometry (UHRMS) was successfully applied to the composition characterization of thiophene compounds in both a low boiling petroleum fraction and a whole crude oil. APCI coupled with trapped ion mobility spectrometry (TIMS) was developed to determine the shape and size of thiophene compounds, providing configuration information that affects the steric hindrance and diffusion behavior of reactants in the desulfurization reaction, which has not been previously reported. Moreover, the comprehensive experimental structural data, expressed as the collision cross section (CCS) of the ions as surrogates of molecules, provided clues to the factors affecting the desulfurization reactivity of thiophene compounds. Further exploration showed that not only qualitative analysis of thiophene compounds can be achieved from the correlation between m/z and CCS, but also molecular size was found to be correlated with CCS that can be used as structural analysis. Overall, the molecular composition and dimension analysis together can provide substantial information for the desulfurization activity of thiophene compounds, facilitating the desulfurization process studies and catalyst design.