Quaterthiophene is used as a fluorescent marker for biological applications, but the intrinsic excited state dynamics for its high triplet-formation yield are still under debate due to the complexity of the molecule structure and the undetermined energy level order. In this work, ultrafast geometry relaxation and intersystem crossing of quaterthiophene in 1,4-dioxane are studied by femtosecond time-resolved spectroscopy combined with quantum calculations. Transient absorption spectra at a pump wavelength of 400 nm are completely recorded up to the delay time of 1 ns. The kinetic traces of excited state absorption indicate that geometry relaxation occurs on the S1 potential energy surface with a time constant of ∼70 ps. Two triplet-triplet absorption bands centered at 563 nm and 600 nm show a direct dynamical conversion. The intersystem crossing is determined to be ∼398 ps. The high triplet yield is measured as ∼0.7 via the efficient intersystem crossing. On the basis of quantum chemical calculations, a general mechanism is proposed to describe the geometry relaxation and intersystem crossing processes.