This study aimed to provide preliminary evidence for the efficacy of invasive laser acupuncture (ILA) for chronic non-specific low back pain (CNLBP). This was a single-center, randomized, patient and assessor-blinded, placebo-controlled, parallel-arm, clinical trial with a 1:1:1 allocation ratio that included a full analysis set. Forty-five participants with CNLBP were randomly assigned to the control group (sham laser), 650 group (650 nm-wavelength ILA), or 830 group (830 nm-wavelength ILA) (n = 15/group). All participants received ILA for 10 min, followed by electroacupuncture for 10 min on the same day. The treatment was performed once per day, twice per week for 4 weeks at bilateral BL23, BL24, BL25, and GB30. The primary outcome was the among-group difference of changes in the visual analog scale (VAS) scores at intervention endpoint (week 4). The secondary outcomes were the among-group difference of changes in VAS at 4 weeks after intervention completion (week 8), those in the Korean version of the Oswestry Disability Index (ODI) and the European Quality of Life Five-Dimension- Five-Level (EQ-5D-5L) at intervention endpoint (week 4) and 4 weeks after intervention completion (week 8). The VAS scores of the 650 group decreased significantly compared with those of the control group (p = 0.047; week 4 vs. week 0). The ODI scores of the 650 group (p = 0.018, week 4 vs. week 0; p = 0.006, week 8 vs. week 0) and 830 group (p = 0.014, week 4 vs. week 0) decreased significantly compared with those of the control group. There was no adverse event related to ILA and no significant difference in changes in vital signs among the three groups. The 650 group showed significant improvements in pain intensity and functional disability. The 830 group showed significant improvements in functional disability. Therefore, ILA therapy at 650 nm and 830 nm wavelengths can be used to treat CNLBP.