To realize highly sensitive immunoassays, high optical density probes conjugated with antibodies for target antigens have been demanded in order to increase the visibility of antigen-antibody complex formation. We herein demonstrate the development of an immunoassay system using magnetic and fluorescent Janus particles as probes in conjunction with an antibody-immobilized microfluidic device. The concentration of the detection limit at which there was a significant difference between SARS-CoV-2 and human coronavirus 229E antigens was 3.1 ng/mL, and the standard deviation of the signal was less than 5%. The immunofluorescent probe and immunoassay system developed in this study are expected to be applicable not only to SARS-CoV-2 but also to the quantitative measurement of various other disease marker proteins and biomolecules.