Abstract: In two-dimensional layered materials, the twist angle has been considered to provide a new degree of freedom for tuning nonlinear optical responses. To investigate the modulation mechanism of the twist angle on second harmonic generation (SHG) in supertwisted helical WS₂ structures, supertwisted helical WS₂ was synthesized on conical substrates formed by tungsten oxide particles via a water-assisted chemical vapor deposition method. The morphology and layer thickness were characterized by atomic force microscopy (AFM), and the evolution of the twist angle and SHG intensity with the number of layers was systematically analyzed in combination with point-by-point SHG measurements in reflection mode. The results showed that for conventional helical structures with a twist angle of 0°, the SHG intensity was significantly enhanced with increasing layer number due to the breaking of central inversion symmetry, whereas for supertwisted helical structures with twist angles of 15° and 20°, the SHG intensity was found to first increase and then decrease with increasing layer number. Further analysis indicated that when the cumulative interlayer twist angle was less than 60°, the inversion symmetry breaking persisted, by which the SHG signal was enhanced; when the cumulative twist angle reached or exceeded 60°, the local inversion symmetry was restored, and the SHG signal was attenuated. The intrinsic relationship among substrate geometry, twist angle, and nonlinear optical response was elucidated in this study, by which an experimental basis was provided for symmetry tuning in two-dimensional twisted systems and for the design of nonlinear photonic devices.