We developed a wash-free biosensor enabling quantitative analysis of cardiac troponin I (cTnI) using retroreflective Janus particle (RJP) as an optical probe. The RJP, we developed, had a metal layer which composed of a high refractive metal in half of the surface to induce the retroreflective phenomenon. Because of the hemisphere of the metal layer, the RJPs were sedimented and twinkled in biosensing microchannel. By using these two properties of RJP, the wash-free optical immunosensor was developed. The performance of the developed sensor was demonstrated via quantitative analysis of serum cTnI.