Semipolar III-nitrides have attracted increasing attention in applications of optoelectronic devices due to the much reduced polarization field. A high-quality semipolar AlN template is the building block of semipolar AlGaN-based deep-ultraviolet light emitting diodes (DUV LEDs), and thus deserves special attention. In this work, a multi-step in situ interface modification technique is developed for the first time, to our knowledge, to achieve high-quality semipolar AlN templates. The stacking faults were efficiently blocked due to the modification of atomic configurations at the related interfaces. Coherently regrown AlGaN layers were obtained on the in situ treated AlN template, and stacking faults were eliminated in the post-grown AlGaN layers. The strains between AlGaN layers were relaxed through a dislocation glide in the basal plane and misfit dislocations at the heterointerfaces. In contrast, high-temperature ex situ annealing shows great improvement in defect annihilation, yet suffers from severe lattice distortion with strong compressive strain in the AlN template, which is unfavorable to the post-grown AlGaN layers. The strong enhancement of luminous intensity is achieved in in situ treated AlGaN DUV LEDs. The in situ interface modification technique proposed in this work is proven to be an efficient method for the preparation of high-quality semipolar AlN, showing great potential towards the realization of high-efficiency optoelectronic devices.