The present invention relates to a method for the in vitro large-scale expansion of double-negative T cells rich in Tscm and Tem. Specifically, the method comprises the step of: (a) providing an initial sample I of peripheral blood obtained from a donor, (b) pretreating the initial sample I to obtain sample II, and (c) culturing sample II in a culture system containing a culture medium suitable for the growth of DNT cell, thereby obtaining a sample III. The expansion process of the present invention is simple and stable, and can achieve the in vitro large-scale culturing of DNT cells rich in Tscm and Tcm cell characteristics. The purity of the final product DNT cells (CD3+CD4CD8) is 85%, which meets the requirements for clinical use.