The 304 stainless steel with a thickness of 1.5 mm is drilled in femtosecond laser (235 fs, 100 kHz) under different defocusing amounts. The drilling rates were recorded. The diameter and taper of micro-holes under different defocusing amounts were measured and calculated. Also, the quality and element distribution of micro-hole sidewall were characterized by optical microscopy and SEM. Experimental results demonstrate that when the defocusing amount deviates from -1.0 mm to 1.0 mm, inlet and outlet diameter of the micro-holes increase, but taper of the micro-holes decreases first and then increases, and the largest taper is 3.32° . Meanwhile, the outlet quality of micro-holes becomes worse gradually. No recast layer or micro-cracks is observed on the sidewall of all micro-holes, while the quality of micro-holes sidewall under negative defocusing amount is better. The element distribution of base metal nearby the sidewall of micro-hole is almost not variable. Compared to positive defocus, the drilling efficiency under negative defocusing amount is higher , and it is about 27 s.