The invention discloses a preparation method of a tapered fiber SERS probe. The preparation method comprises: plating a tapered fiber surface of a tapered fiber structure with a noble metal nano film;and coupling femtosecond laser to a tapered optical fiber plated with the noble metal nano film by using a laser coupling unit, wherein the femtosecond laser transmitted in the tapered fiber structure interacts with the noble metal nano film coating the tapered fiber surface; the noble metal nano film absorbs the laser energy of the femtosecond laser and converts the same into heat of the noble metal nano film; the noble metal nano film undergoes an adiabatic high temperature annealing process to form dispersing noble metal nano particles on the tapered fiber surface, thus achieving the preparation of the fiber SERS probe. The preparation method by femtosecond laser, provided by the invention can solve the problem of photothermal accumulation or damage confronted by the existing continuous light preparation of a fiber SERS probe; and solves the problem that it is difficult for the existing femtosecond laser to prepare a tapered fiber SERS probe by coupling the femtosecond laser into the fiber for transmission.