Abstract: The disclosure discloses a method for preparing a multisubunit SCF E3 ligase with a fusion protein through in vitro reconstitution, and a use of the multisubunit SCF E3 ligase. The method for preparing a multisubunit SCF E3 ligase with a fusion protein through in vitro reconstitution disclosed by the disclsoure includes: subjecting an engineered SKP1-Cullin1-RBX1 fusion protein (referred to as eSCR) to a reaction with an F-box protein in a reaction system to obtain the multisubunit SCF E3 ligase, where the eSCR fusion protein has an amino acid sequence from position 10 to position 1062 of SEQ ID NO: 2. Experimental results show that different multisubunit SCF E3 ligases are successfully prepared with the eSCR fusion protein through in vitro reconstitution in the disclsoure; the reconstituted multisubunit E3 ligase has biological activity.