We report the design and development of an optical fiber probe-based Helium–Neon (He–Ne) low-level laser therapy system for tissue regeneration. Full thickness excision wounds on Swiss albino mice of diameter 15 mm were exposed to various laser doses of 1, 2, 3, 4, 6, 8 and 10 J cm−2 of the system with appropriate controls, and 2 J cm−2 showing optimum healing was selected. The treatment schedule for applying the selected laser dose was also standardized by irradiating the wounds at different postwounding times (0, 24 and 48 h). The tissue regeneration potential was evaluated by monitoring the progression of wound contraction and mean wound healing time along with the hydroxyproline and glucosamine estimation on wound ground tissues. The wounds exposed to 2 J cm−2 immediately after wounding showed considerable contraction on days 5, 9, 12, 14, 16 and 19 of postirradiation compared with the controls and other treatment schedules, showing significant (P < 0.001) decrease in the healing time. A significant increase in hydroxyproline and glucosamine levels was observed for the 2 J cm−2 irradiation group compared with the controls and other treatment groups. In conclusion, the wounds treated with 2 J cm−2 immediately after the wounding show better healing compared with the controls.