Inflammation of synovial membrane and degeneration of articular cartilage in osteoarthritis (OA) lead to major changes in joint space width (JSW) and biochemical components such as collagen-II telopeptide (CTX-II) and matrix metallo protineases (MMP-3, 8, and 13). Low-level laser therapy (LLLT) is thought to have an analgesic effect as well as biomodulatory effect on microcirculation and cartilage regeneration in animal studies. The objective of this study was to examine the analgesic and biochemical effect of LLLT in patients with knee osteoarthritis. Subjects (n = 34) who fulfilled the selection criteria were randomly divided into active group (n = 17) and placebo group. Subjects in active group were irradiated laser with the frequency of 3 days per week for 4 weeks with the specific parameters on 8 different points on the joint at 1.5 J per point for 60 s for 8 points for a total dose of 12 J in a skin contact method. The placebo group was treated with the same probe with minimum emission of energy. Visual analog scale for pain intensity, joint space width, collagen-II telopeptide, and matrix metallo protinease-3, 8, and 13 was measured before treatment and at 4 and 8 weeks following treatment. Data are analyzed with mean values and standard deviation with p < 0.05. Baseline values of all outcome measures show insignificant difference (p > 0.05) in both groups which shows homogeneity. After 4- and 8-week treatment, active laser group shows more significant difference (p < 0.001) in all the parameters than the placebo laser group (p > 0.05). Our results show that low-level laser therapy was more efficient in reducing pain and improving cartilage thickness through biochemical changes.