The invention provides a preparation method of reconstituted tobacco. The preparation method comprises the following steps of: 1, uniformly mixing tobacco stems, tobacco powder and water, then adding a cell disruption enzyme and an assistant I for carrying out enzymatic hydrolysis; 2, heating and extracting a mixture in the step 1, carrying out enzyme deactivation, then filtering to obtain filtrate and filter residue, concentrating the filtrate to obtain a concentrated solution; 3, adding a mixed culture and an assistant II in the concentrated solution, carrying out fermentation and enzymatic hydrolysis to obtain a tobacco extraction solution; 4, adding slurry in the filter residue in the step 1, uniformly mixing, adding hydrogen peroxide for carrying out oxygenolysis, then adding catalase for hydrolyzing residual peroxide to obtain mixed slurry; and 5, preparing the mixed slurry into a slice paper base, coating the tobacco extraction solution on the slice paper base, drying and preparing into the reconstituted tobacco. According to the preparation method of the reconstituted tobacco, the mixed culture is fermented in the concentrated solution, and thus the content of lignin can be reduced, and the smoking effect is improved.