The invention discloses a method used for evaluating an immunomodulatory effect of lactobacillus acidophilus and an application of the method, and belongs to the field of biotechnology. According to the method provided by the invention, a dynamic change situation of immunity-related differentially expressed genes is detected, wherein the differentially expressed genes include up-regulated immunity-related genes CCL2, CCL20, IL1 alpha, IL1 beta, IL6ST, IL8, PTX3 and TNFRSF9 and a down-regulated immunity-related gene CXCR4. Through the method, the immunomodulatory effect of the lactobacillus acidophilus can be evaluated only by detecting nine immunity-related genes, and quick detection can be realized by utilizing Real Time PCR, so that the time is greatly shortened and the deficiency of existing evaluation of the immunomodulatory effect of the lactobacillus acidophilus is overcome; and the method is well verified in a mouse test and a Caco-2 cell.