In this work, the fabrication of gradient periodic line-like structures with continuously changing spatial period on stainless steel by means of two-beam direct laser interference patterning (DLIP) is studied. A 70 ps (picosecond [ps]) pulsed laser operating at 532 nm wavelength and 0.64 J cm ⁻² laser fluence is used to produce the gradient microstructures. By continuously changing the overlapping angle between the sub-beams, the spatial period of gradient structures smoothly increases from 2.01 ± 0.09 to 3.99 ± 0.12 μm within a 500 μm long processed area. Different pulse-to-pulse overlaps are applied to study the influence of cumulated fluence on the topography of gradient structures. The surface topography is analyzed by optical confocal microscopy and is characterized by the roughness parameters R _a and R _z. Fourier transformations of the confocal images allows the verification of the variations of the spatial periods. In the experiments performed in this work, design rules are provided for fabricating complex microstructures in the future using DLIP-based technology to achieve advanced surface function like anisotropic wetting or cell growth.