In this study, the radiative and nonradiative decay pathways from the first singlet excited states (denoted as S1) of three bithiophene-fused isoquinolines were investigated by using the mixed-reference spin-flip time-dependent density functional theory approach. These isoquinolines, which are prepared via [2 + 2 + 2] cycloaddition reactions between three types of bithiophene-linked diynes and nitriles, exhibit different fluorescence quantum yields in response to the positions of their sulfur atoms. The decay processes, including the fluorescence emission and internal conversion, were considered. In the internal conversion pathway, the minimum energy conical intersection structures between the ground and first singlet excited states (denoted as S0/S1 MECI) of the ring strain for the isoquinoline skeleton and the ring opening of the thiophene skeleton were systematically explored. Dewar-type ring strain resulted in the smallest energy barrier from the equilibrium geometries of the ground state (denoted as S0) to the MECI structures between the S0 and S1 states. The energy difference between the three types of bithiophene-fused isoquinolines at the transition state geometries of the S1 state varies owing to the steric effects between the methyl groups and the hydrogen atom of the thiophene ring, and the excitation energy increases owing to a decrease in aromaticity. In addition, the oscillator strengths of the S0 and S1 states were evaluated at the equilibrium geometries of the S1 state to determine the contribution of the fluorescence process. The obtained theoretical results are consistent with the experimental results.