The present invention provides a method for detecting an antigen in an analysis sample, the method including: (a) contacting an analysis sample with a detection antibody with which a marker generating a detectable signal is combined and which specifically binds to the antigen; (b) contacting a capture antibody with the resultant product of step (a), the capture antibody specifically binding to an antigen to be detected; (c) contacting the detection antibody, with which the marker generating a detectable signal is combined, with a reference substance which is bound to a surface of a solid substrate and which includes an epitope to which the detection antibody specifically binds; (d) measuring signals generated from the markers of the resultant product of step (b) and the resultant product of step (c); and (e) analyzing the measured signals to determine the presence or absence and amount of the antigen in the analysis sample. The method for detecting an antigen of the present invention can control the flow and the reaction time of an analysis sample, thereby improving sensitivity and minimizing the influences by the concentration of the analysis sample or the temperature of the detection reaction, and thus improving stability, reliability, and reproducibility of data, when compared with the conventional method for detecting an antigen. Accordingly, the method and apparatus for detecting an antigen of the present invention can be easily operated without specialized skills, thereby instantly obtaining the presence or absence and amount of detection antigen in the analysis sample through on-site diagnosis.