The invention discloses a preparation method of flammulina velutipes dietary fiber. Flammulina velutipes dietary fiber leftovers or waste is subjected to raw material pretreatment with an alkaline process in combination with a fermentation method, then washed, filtered and bleached, and the flammulina velutipes dietary fiber is obtained. The preparation method has the benefits as follows: the dietary fiber obtained with the preparation method has relatively high purity and high extraction rate, the preparation method is simple and easy to implement, the technological conditions are easy to control, the production cost is low, and large-scale industrial production is easy to realize; the production cost is reduced, resource waste is reduced, and the additional value of flammulina velutipes is increased; the product has high water holding power and expansibility, has good physiological activity, is in light yellow, does not taste rough and can be applied to cakes and pastries, biscuits, bread and other kinds of food as a better food additive, and a new approach is provided for comprehensive utilization of processing by-products of flammulina velutipes.