The present invention discloses a method for simultaneous detection of cancer-associated fibroblasts and expressed proteins thereof, belonging to the technical field of tissue immunofluorescence. According to the method, a first type of quantum dot is used for labeling the antibody of a cell marker of the cancer-associated fibroblasts, or labeling a secondary antibody capable of specific binding to the antibody of the cell marker of the cancer-associated fibroblasts, or labeling streptavidin, and at least a second type of quantum dot is used for labeling the antibody of an expressed protein of the cancer-associated fibroblasts, or labeling a secondary antibody capable of specific binding to the antibody of the expressed protein of the cancer-associated fibroblasts, or labelling streptavidin; wherein the emission wavelengths of the first type of quantum dot and the second type of quantum dot are different from each other, and the first type of quantum dot and the second type of quantum dot does not label the streptavidin simultaneously. The invention also discloses a kit for simultaneous detection of cancer-associated fibroblasts and expressed protein thereof. The method of the invention is simple, efficient, accurate, and simultaneous in detection, suitable for promotion to the simultaneous labeling and quantitative analysis of a variety of other cancer-associated markers.