This in‐vitro study aimed to explore the potential of Diode Laser by quantifying the release of transforming growth factor‐beta 1 (TGF‐β1), platelet‐derived growth factor (PDGF)‐BB, and vascular endothelial growth factor (VEGF) from root dentin after ethylenediaminetetraacetic acid (EDTA) and diode laser. In 30 hemi‐roots distributed into five groups: G1: Tris‐buffered solution (TBS); G2: 17%EDTA; G3: 17% EDTA +650 nm Diode Laser; G4:17% EDTA +810 nm Diode Laser; and G5: 810 nm Diode Laser. The concentration of the three factors was quantified using a cytokine bead array. Statistical tests were performed to estimate intergroup differences (p ≤ 0.05). TGF‐β1 and VEGF were solubilised in all test protocols. The ability of low‐level power diode lasers to release proteins from the matrix is limited. Its effect on the release of VEGF and PDGF‐BB does not make a difference. A synergy between EDTA and Diode Laser led to a greater proportion of TGF‐β1 release.