The invention discloses a method for establishing a tomato nuclear male sterile line with a green stem marker, which comprises the following steps: simultaneously editing a fertility gene SlMs1035 ina receptor tomato genome and a linked marker gene SlF3H thereof by using a CRISPR/Cas9 system so as to cause the two genes to lose functions, and the tomato material obtained with the SlMs1035 and theSlF3H simultaneously losing function through ridding the transgene structure by carrying out selfing or hybridization is the tomato nuclear male sterile line with the green stem mark; wherein the marker gene SlF3H is an anthocyanin synthesis gene. As the stem of the sterile plants is green at the seedling stage, the sterile plants can be selected before the plants blossom. The method for establishing tomato nuclear male sterile line with the green stem marker adopts a molecular design breeding method to obtain a tomato nuclear male sterile line with a non-transgenic seedling stage marker character, and can be applied to transforming a tomato hybrid female parent into a nuclear sterile line with a green stem marker for tomato seed production through hybrid.