The invention relates to a method for preparing a super-hydrophilic and super-hydrophobic composite SERS substrate based on femtosecond laser, and belongs to the technical field of laser application.According to the method, the combination of the super-hydrophobic surface and the hydrophilic substrate is realized by adopting femtosecond laser selective deposition, the super-hydrophilic region isselectively processed through laser direct writing, and the preparation of the patterned super-hydrophilic and super-hydrophobic alternated surface is realized. According to the preparation method, only femtosecond laser is needed, a sample does not need to be moved, and the preparation method is suitable for any intrinsic hydrophilic substrate. The preparation method of the super-hydrophilic andsuper-hydrophobic composite SERS substrate provides a simple, efficient, flexible and widely-applicable processing scheme for preparation of the super-hydrophilic and super-hydrophobic composite SERSsubstrate. In addition, the auxiliary heating is carried out in the liquid drop concentration process, and liquid drops containing target molecules and AU nano-particles are completely concentrated tothe super-hydrophilic region by utilizing a water vapor membrane, so that the concentration area is controllable so as to output a stable Raman detection signal.