This paper reports on the fabrication of marking units of controllable size ranging from 300 to 500 μm equipped with anti-reflective micro-structures using defocused femtosecond laser on titanium alloy TC4. The results show that the range of diameters of marking units (laser ablation-based craters) goes through three stages with the increase of the pulse number. The craters are too shallow for the first stage and too deep for the third stage to meet the criterion of depth, so the second stage turns out to be the optimal stage of parameter selection, where the diameter and depth of marking units are within a desired range with modification of laser energy and defocusing amount. Besides meeting the marking requirements of the size and morphology, the anti-reflective micro-structures with great color difference are formed on the surface of marking units, which contributes to the high recognition rate. Compared with the conventional marking methods, this method has a great recognition rate without recast layer and micro-cracks. Therefore, the femtosecond laser-based processing would provide a new marking technology with high efficiency and quality. ? 2019 Elsevier Ltd