The invention discloses a preparation method of a barley extract fermented by lactobacillus and an anti-tumor effect of the barley extract fermented by lactobacillus, belonging to the field of food biotechnology. Lactobacillus plantarum (lactobacillus plantarum Dy-1, CGMCC No.6016) is screened from pickled vegetables to serve as a fermentation strain for fermenting the barley, and fermentation conditions are controlled, so that the extract of fermented barley is obtained. Cell tests indicate that the extract has obvious effects on promoting apoptosis and suppressing multiplication of human colon cancer HT-29 cells and gastric cancer SGC-7901 cells, and a model test of the nude mouse bearing the tumor with the human colon cancer HT-29 indicates that the extract has obvious effects on suppressing tumor growth and improving the immune activity of the nude mouse bearing the tumor. According to the preparation method of the barley extract fermented by lactobacillus, the barley is pretreated simply and the production cost is low; and direct vat set fermentation is employed, so that the preparation method of the barley extract fermented by lactobacillus is convenient and rapid, safe and stable, and convenient to control the quality.