Evidence strongly supports that low-level laser therapy (LLLT) is an effective physical modality for the treatment of pain associated with myofascial trigger points (MTrP). However, the effect of laser fluence (energy intensity in J/cm²) on biochemical regulation related to pain is unclear. To better understand the biochemical mechanisms modulated by high- and low-fluence LLLT at myofascial trigger spots (MTrSs; similar to human MTrPs) in skeletal muscles of rabbits, the levels of β-endorphin (β-ep), substance P (SP), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) were investigated in this study. New Zealand rabbits (2.5–3.0 kg in weight) were used in this study. High-fluence LLLT (27 J/cm²), low-fluence LLLT (4.5 J/cm²), or sham operations were applied on MTrSs of biceps femoris of rabbits for five sessions (one session per day). Effects of LLLT at two different fluences on biceps femoris, dorsal root ganglion (DRG), and serum were determined by β-ep, SP, TNF-α, and COX-2 immunoassays. LLLT irradiation with fluences of 4.5 and 27 J/cm² at MTrSs can significantly reduce SP level in DRG. LLLT with lower fluence of 4.5 J/cm² exerted lower levels of TNF-α and COX-2 expression in laser-treated muscle, but LLLT with higher fluence of 27 J/cm² elevated the levels of β-ep in serum, DRG, and muscle. This study demonstrated fluence-dependent biochemical effects of LLLT in an animal model on management of myofascial pain. The findings can contribute to the development of dosage guideline for LLLT for treating MTrP-induced pain.