Despite the literature providing compelling evidence for the medical efficacy of photobiomodulation (PBM) therapy, its consistency in terms of accuracy and standardization needs improving. Identification of new technology and reliable and ethical biological models is, therefore, a challenge for researchers working on PBM. We tested the reliability of PBM irradiation through a novel delivery probe with a flat-top beam profile on the regenerating amphioxus Branchiostoma lanceolatum. The caudalmost 9 ± 1 myotomes, posterior to the anus, were excised using a sterile lancet. Animals were randomly split into three experimental groups. In the control group, the beam area was bounded with the 635-nm red-light pointer (negligible power, <0.5 mW) and the laser device was coded to irradiate 810 nm and 0 W. In Group laser-1, the beam area was bounded with the same 635-nm red-light pointer and irradiated at 810 nm, 1 W in CW for 60 s, spot-size 1 cm2, 1 W/cm2, 60 J/cm2, and 60 J; irradiation was performed every day for two weeks. In Group laser-2, the beam area was bounded with the same 635-nm red-light pointer and irradiated at 810 nm, 1 W in CW for 60 s, spot-size 1 cm2, 1 W/cm2, 60 J/cm2, and 60 J; irradiation was performed on alternate days for four weeks. We observed that PBM improved the natural wound-healing and regeneration process. The effect was particularly evident for the notochord. Daily irradiation better supported the regenerative process.