The invention provides an immunoassay based on a carbon nanomaterial (carbon spot), comprising the following steps of: step A, preparing a carbon spot, and marking an immunoreagent antigen or antibody on a surface of the carbon spot so that the carbon spot becomes an immunoreagent-marked carbon spot; step B, mixing the immunoreagent-marked carbon spot, a sample to be tested and a solid phase substance coated by the immunoreagent antigen or antibody to complete immunoreactions, thus forming an immune complex, and separating the immune complex from a free immunoreagent; and step C, adding an oxidant to the immune complex for chemiluminescence detection, and/or directly enabling the immune complex to be subjected to fluorescence detection. According the immunoassay based on the carbon nanomaterial, the chemiluminescence detection and the fluorescence detection can be performed by using the carbon nanomaterial, and the optical stability is good.